Konstantina M. Stankovic, MD, PhD, FACS

All Publications

• An In Vitro Oxidative Stress Model of the Human Inner Ear Using Human-Induced Pluripotent Stem Cell-Derived Otic Progenitor Cells. Antioxidants (Basel, Switzerland) Jeong, M., Kurihara, S., Stankovic, K. M. 2024; 13 (11)

  Abstract

  The inner ear organs responsible for hearing (cochlea) and balance (vestibular system) are susceptible to oxidative stress due to the high metabolic demands of their sensorineural cells. Oxidative stress-induced damage to these cells can cause hearing loss or vestibular dysfunction, yet the precise mechanisms remain unclear due to the limitations of animal models and challenges of obtaining living human inner ear tissue. Therefore, we developed an in vitro oxidative stress model of the pre-natal human inner ear using otic progenitor cells (OPCs) derived from human-induced pluripotent stem cells (hiPSCs). OPCs, hiPSCs, and HeLa cells were exposed to hydrogen peroxide or ototoxic drugs (gentamicin and cisplatin) that induce oxidative stress to evaluate subsequent cell viability, cell death, reactive oxygen species (ROS) production, mitochondrial activity, and apoptosis (caspase 3/7 activity). Dose-dependent reductions in OPC cell viability were observed post-exposure, demonstrating their vulnerability to oxidative stress. Notably, gentamicin exposure induced ROS production and cell death in OPCs, but not hiPSCs or HeLa cells. This OPC-based human model effectively simulates oxidative stress conditions in the human inner ear and may be useful for modeling the impact of ototoxicity during early pregnancy or evaluating therapies to prevent cytotoxicity.

  View details for DOI 10.3390/antiox13111407

  View details for PubMedID 39594548

  View details for PubMedCentralID PMC11591063

• Dynamic micro-optical coherence tomography enables structural and metabolic imaging of the mammalian cochlea. Frontiers in molecular neuroscience Schulz-Hildebrandt, H., Spasic, S., Hou, F., Ting, K. C., Batts, S., Tearney, G., Stankovic, K. M. 2024; 17: 1436837

  Abstract

  Sensorineural hearing loss (SNHL) is caused by damage to the mechanosensory hair cells and auditory neurons of the cochlea. The development of imaging tools that can directly visualize or provide functional information about a patient's cochlear cells is critical to identify the pathobiological defect and determine the cells' receptiveness to emerging SNHL treatments. However, the cochlea's small size, embedded location within dense bone, and sensitivity to perturbation have historically precluded high-resolution clinical imaging. Previously, we developed micro-optical coherence tomography (μOCT) as a platform for otologic imaging in animal models and human cochleae. Here we report on advancing μOCT technology to obtain simultaneously acquired and co-localized images of cell viability/metabolic activity through dynamic μOCT (DμOCT) imaging of intracellular motion. DμOCT obtains cross-sectional images of ATP-dependent movement of intracellular organelles and cytoskeletal polymerization by acquiring sequential μOCT images and computing intensity fluctuation frequency metrics on a pixel-wise basis. Using a customized benchtop DμOCT system, we demonstrate the detailed resolution of anatomical and metabolic features of cells within the organ of Corti, via an apical cochleostomy, in freshly-excised adult mouse cochleae. Further, we show that DμOCT is capable of capturing rapid changes in cochlear cell metabolism following an ototoxic insult to induce cell death and actin stabilization. Notably, as few as 6 frames can be used to reconstruct cochlear DμOCT images with sufficient detail to discern individual cells and their metabolic state. Taken together, these results motivate future development of a DμOCT imaging probe for cellular and metabolic diagnosis of SNHL in humans.

  View details for DOI 10.3389/fnmol.2024.1436837

  View details for PubMedID 39449964

  View details for PubMedCentralID PMC11499234

• Human otic progenitor cell models of congenital hearing loss reveal potential pathophysiologic mechanisms of Zika virus and cytomegalovirus infections. mBio Harding, A. T., Ocwieja, K., Jeong, M., Zhang, Y., Leger, V., Jhala, N., Stankovic, K. M., Gehrke, L. 2024: e0019924

  Abstract

  Congenital hearing loss is a common chronic condition affecting children in both developed and developing nations. Viruses correlated with congenital hearing loss include human cytomegalovirus (HCMV) and Zika virus (ZIKV), which causes congenital Zika syndrome. The mechanisms by which HCMV and ZIKV infections cause hearing loss are poorly understood. It is challenging to study human inner ear cells because they are encased in bone and also scarce as autopsy samples. Recent advances in culturing human stem cell-derived otic progenitor cells (OPCs) have allowed us herein to describe successful in vitro infection of OPCs with HCMV and ZIKV, and also to propose potential mechanisms by which each viral infection could affect hearing. We find that ZIKV infection rapidly and significantly induces the expression of type I interferon and interferon-stimulated genes, while OPC viability declines, at least in part, from apoptosis. In contrast, HCMV infection did not appear to upregulate interferons or cause a reduction in cell viability, and instead disrupted expression of key genes and pathways associated with inner ear development and function, including Cochlin, nerve growth factor receptor, SRY-box transcription factor 11, and transforming growth factor-beta signaling. These findings suggest that ZIKV and HCMV infections cause congenital hearing loss through distinct pathways, that is, by inducing progenitor cell death in the case of ZIKV infection, and by disruption of critical developmental pathways in the case of HCMV infection.Congenital virus infections inflict substantial morbidity and devastating disease in neonates worldwide, and hearing loss is a common outcome. It has been difficult to study viral infections of the human hearing apparatus because it is embedded in the temporal bone of the skull. Recent technological advances permit the differentiation of otic progenitor cells (OPCs) from human-induced pluripotent stem cells. This paper is important for demonstrating that inner ear virus infections can be modeled in vitro using OPCs. We infected OPCs with two viruses associated with congenital hearing loss: human cytomegalovirus (HCMV), a DNA virus, or Zika virus (ZIKV), an RNA virus. An important result is that the gene expression and cytokine production profiles of HCMV/ZIKV-infected OPCs are markedly dissimilar, suggesting that mechanisms of hearing loss are also distinct. The specific molecular regulatory pathways identified in this work could suggest important targets for therapeutics.

  View details for DOI 10.1128/mbio.00199-24

  View details for PubMedID 38440980

• Correction: Natarajan et al. Noise-Induced Hearing Loss. J. Clin. Med. 2023, 12, 2347. Journal of clinical medicine Natarajan, N., Batts, S., Stankovic, K. M. 2024; 13 (4)

  Abstract

  Error in Table [...].

  View details for DOI 10.3390/jcm13040944

  View details for PubMedID 38398496

  View details for PubMedCentralID PMC10889190

• Surgical Considerations in Inner Ear Gene Therapy from Human Temporal Bone Anatomy. The Laryngoscope Brown, A., Zhu, M., Rohani, A., Ladak, H., Agrawal, S. K., Stankovic, K. M., Welling, D. B. 2024

  Abstract

  OBJECTIVE(S): Recently directed methods of inner ear drug delivery underscore the necessity for understanding critical anatomical dimensions. This study examines anatomical measurements of the human middle and inner ear relevant for inner ear drug delivery studied with three different imaging modalities.METHODS: Post-mortem human temporal bones were analyzed using human temporal bone histopathology (N=24), micro computerized tomography (muCT; N=4), and synchrotron radiation phase-contrast imaging (SR-PCI; N=7). Nine measurements involving the oval and round windows were performed when relevant anatomical structures were visualized for subsequent age-controlled analysis, and comparisons were made between imaging methods.RESULTS: Combined human temporal bone histopathology showed the mean distance to the saccule from the center of the stapes footplate (FP) was 2.07±0.357mm and the minimum distance was 1.23mm. The mean distance from the round window membrane (RWM) to the osseous spiral lamina (OSL) was 1.75±0.199mm and the minimum distance was 1.43mm. Instruments inserted up to 1mm past the center of the FP are unlikely to cause saccular damage, provided there are no endolymphatic hydrops. Similarly, instruments inserted up to 1mm through the RWM in the trajectory toward the OSL are unlikely to cause OSL damage.CONCLUSION: The combined analyses of inner-ear dimensions of age-controlled groups and imaging modalities demonstrate critical dimensions of importance to consider when inserting delivery vehicles into the human cochlea.LEVEL OF EVIDENCE: N/A Laryngoscope, 2024.

  View details for DOI 10.1002/lary.31259

  View details for PubMedID 38197496

• Tinnitus prevalence, associated characteristics, and related healthcare use in the United States: a population-level analysis. Lancet regional health. Americas Batts, S., Stankovic, K. M. 2024; 29: 100659

  Abstract

  Tinnitus is a potentially disabling condition with few treatments. We examined the prevalence and characteristics of tinnitus among demographic groups in the United States (US) and assessed associated factors and tinnitus-related healthcare.We included adults with and without bothersome tinnitus from the nationally representative 2014 National Health Interview Survey (NHIS; raw n = 36,697), the latest year with tinnitus data. We evaluated tinnitus prevalence and characteristics (frequency, severity, duration) overall and among groups defined by sex and race/ethnicity. Logistic regression with adjusted Wald tests were used for comparisons in NHIS-weighted populations by sex and race/ethnicity, and to evaluate associations between demographic/medical characteristics and noise exposure on tinnitus risk.The US prevalence of tinnitus was 11.2% (95% CI: 10.8%, 11.7%; ∼27 million people) in 2014. Of those with tinnitus, 41.2% always had symptoms and 28.3% had ≥15 years symptom duration; the rates were significantly higher among men vs. women and non-Hispanic (nHW) vs. Hispanic Whites (HW), Blacks, or other ethnicity. Significantly more women vs. men and HW vs. nHW reported severe tinnitus. Sex and race/ethnicity, except Asian, were not significantly associated with tinnitus when age, otologic/medical disorders, and noise exposure were included in the model. Significantly lower rates of all minority groups discussed tinnitus with a doctor compared to nHW, and among those who did, Blacks were significantly less likely to receive tinnitus evaluation than nHWs.Tinnitus prevalence varies across US demographic groups and racial differences were identified in the delivery of tinnitus-related healthcare.Rich Robbins, Bertarelli Foundation Endowed Professorship.

  View details for DOI 10.1016/j.lana.2023.100659

  View details for PubMedID 38269207

  View details for PubMedCentralID PMC10806285

• Genetic Discovery Enabled by A Large Language Model. bioRxiv : the preprint server for biology Tu, T., Fang, Z., Cheng, Z., Spasic, S., Palepu, A., Stankovic, K. M., Natarajan, V., Peltz, G. 2023

  Abstract

  Artificial intelligence (AI) has been used in many areas of medicine, and recently large language models (LLMs) have shown potential utility for clinical applications. However, since we do not know if the use of LLMs can accelerate the pace of genetic discovery, we used data generated from mouse genetic models to investigate this possibility. We examined whether a recently developed specialized LLM (Med-PaLM 2) could analyze sets of candidate genes generated from analysis of murine models of biomedical traits. In response to free-text input, Med-PaLM 2 correctly identified the murine genes that contained experimentally verified causative genetic factors for six biomedical traits, which included susceptibility to diabetes and cataracts. Med-PaLM 2 was also able to analyze a list of genes with high impact alleles, which were identified by comparative analysis of murine genomic sequence data, and it identified a causative murine genetic factor for spontaneous hearing loss. Based upon this Med-PaLM 2 finding, a novel bigenic model for susceptibility to spontaneous hearing loss was developed. These results demonstrate Med-PaLM 2 can analyze gene-phenotype relationships and generate novel hypotheses, which can facilitate genetic discovery.

  View details for DOI 10.1101/2023.11.09.566468

  View details for PubMedID 37986848

• Identification of immune-related candidate biomarkers in plasma of patients with sporadic vestibular schwannoma. Science advances Vasilijic, S., Atai, N. A., Hyakusoku, H., Worthington, S., Ren, Y., Sagers, J. E., Sahin, M. I., Brown, A., Reddy, R., Malhotra, C., Fujita, T., Landegger, L. D., Lewis, R., Welling, D. B., Stankovic, K. M. 2023; 9 (45): eadf7295

  Abstract

  Vestibular schwannoma (VS) is an intracranial tumor arising from neoplastic Schwann cells and typically presenting with hearing loss. The traditional belief that hearing deficit is caused by physical expansion of the VS, compressing the auditory nerve, does not explain the common clinical finding that patients with small tumors can have profound hearing loss, suggesting that tumor-secreted factors could influence hearing ability in VS patients. We conducted profiling of patients' plasma for 66 immune-related factors in patients with sporadic VS (N > 170) and identified and validated candidate biomarkers associated with tumor size (S100B) and hearing (MCP-3). We further identified a nine-biomarker panel (TNR-R2, MIF, CD30, MCP-3, IL-2R, BLC, TWEAK, eotaxin, and S100B) with outstanding discriminatory ability for VS. These findings revealed possible therapeutic targets for VS, providing a unique diagnostic tool that may predict hearing change and tumor growth in VS patients, and may inform the timing of tumor resection to preserve hearing.

  View details for DOI 10.1126/sciadv.adf7295

  View details for PubMedID 37948527

  View details for PubMedCentralID PMC10637750

• Promoting Surgeon-Scientists in Otolaryngology-Head and Neck Surgery-From Bench to Bedside. JAMA otolaryngology-- head & neck surgery Kosaraju, N., Keating, D. P., Kim, G. S., Moore, L. S., Stankovic, K. M. 2023

  Abstract

  Surgeon-scientists (defined as principal investigators [PIs] with a Doctor of Medicine [MD] degree or a combined MD and Doctor of Philosophy [PhD] degree) in otolaryngology-head and neck surgery (OHNS) are imperative for achieving clinical translation in the OHNS field.To (1) raise awareness about the current state of surgeon-scientists in OHNS, (2) contextualize the landscape of surgeon-scientists in OHNS by comparing it to those of neurosurgery and ophthalmology, and (3) identify strategies for attracting and retaining surgeon-scientists in OHNS.Research funding data from fiscal years 2015 to 2021 among surgeon-scientists in OHNS, neurosurgery, and ophthalmology were obtained from the National Institutes of Health (NIH) Research Portfolio Online Reporting Tools Expenditures and Results and the US Department of Defense (DOD) Congressionally Directed Medical Research Programs awards database. The Association of American Medical Colleges provided the total number of active physicians in each specialty per year and the number and percentage of residents with an MD-PhD degree in each specialty per year. Cohen d was used to express the standardized value of the magnitude of the mean difference between compared groups.From 2015 to 2021, on average, there were 9566 active physicians in OHNS, 5559.8 in neurosurgery, and 18908.8 in ophthalmology. In OHNS, a greater number of NIH K (research career development) grants were held by surgeon-scientists than by PIs with a PhD degree (21.4 vs 5.1; mean difference, 16.3; 95% CI, 14.3-18.3; Cohen d = 9.6), whereas most NIH R (research) and U (cooperative agreement) grants (144.1 vs 81.6; mean difference, 62.6; 95% CI, 46.3-78.9; Cohen d = 4.5) and DOD grants (9.9 vs 4.1; mean difference, 5.7; 95% CI, 1.0-10.4; Cohen d = 1.4) were held by PIs with a PhD degree. In a comparison of OHNS to neurosurgery and ophthalmology, after the number of R and U grants was scaled by the number of physicians in each field, neurosurgery had a much greater number of grants per surgeon than OHNS (0.02 vs 0.01; mean difference, 0.01; 95% CI, 0.01-0.02; Cohen d = 4.2). Additionally, neurosurgeons received a much larger R and U grant amount per physician than otolaryngologists ($10 630.20 vs $4511.80; mean difference, $6118.40; 95% CI, $2625.90-$9610.80; Cohen d = 2.0). For the R and U grant metrics, there were no meaningful differences between OHNS and ophthalmology.Results of this database study showed that from 2015 to 2021, the number of governmental grants held by surgeon-scientists in OHNS increased, but there is room for improvement given the metrics of neurosurgeons, a population smaller than otolaryngologists. Possible strategies include intramural research grants, surgeon-scientist training programs, and partnerships between specialty societies and NIH administering institutes and centers.

  View details for DOI 10.1001/jamaoto.2023.3353

  View details for PubMedID 37856105

• Current and Emerging Therapies for Chronic Subjective Tinnitus. Journal of clinical medicine Park, K. W., Kullar, P., Malhotra, C., Stankovic, K. M. 2023; 12 (20)

  Abstract

  Chronic subjective tinnitus, the perception of sound without an external source for longer than six months, may be a greatly debilitating condition for some people, and is associated with psychiatric comorbidities and high healthcare costs. Current treatments are not beneficial for all patients and there is a large need for new therapies for tinnitus.Unlike rarer cases of objective tinnitus, chronic subjective tinnitus often has no obvious etiology and a diverse pathophysiology. In the absence of objective testing, diagnosis is heavily based on clinical assessment. Management strategies include hearing aids, sound masking, tinnitus retraining therapy, cognitive behavioral therapy, and emerging therapies including transcranial magnetic stimulation and electrical stimulation.Although current treatments are limited, emerging diagnostics and treatments provide promising avenues for the management of tinnitus symptoms.

  View details for DOI 10.3390/jcm12206555

  View details for PubMedID 37892692

  View details for PubMedCentralID PMC10607630

• Immune Profiling of Secreted Factors from Human Vestibular Schwannoma Cells and Tumor-associated Macrophages. The Laryngoscope Stankovic, K. M., Batts, S., Welling, D. B., Vasilijic, S. 2023

  Abstract

  This study compared the immune-related secretory capacity of human vestibular schwannoma (VS) and tumor-assisted macrophages (TAMs) with their normal counterparts (Schwann cells [SC] and peripheral blood monocyte-derived macrophages [Mo-MFs], respectively), and examined relationships with presurgical hearing and tumor size.VS tumors (n = 16), auditory nerve (n = 1), blood (n = 9), and great auricular nerves (n = 3) were used. SCs (S100B+ ) and TAMs (CD68+ ) were isolated from VS tissue for culture. The secreted levels of 65 immune-related factors were measured and compared using unpaired t-tests with Welch correction (schwannoma vs. SCs) or Mann-Whitney tests (TAMs and Mo-MFs). Associations between factor concentration and word recognition (WR), pure-tone average (PTA), and tumor size were evaluated with Spearman correlation.Secreted factors with significantly higher concentrations in schwannoma versus SC supernatants included IL-2 and BAFF, whereas MMP-1, IL-6, FGF-2, VEGF-A, MIP-3α, and GRO-α concentrations were significantly higher in TAMs versus Mo-MFs (all p < 0.05). Worse WR was significantly associated with higher secretion of fractalkine, eotaxin-3, CD30, and IL-16 by VS cells; IP-10, eotaxin-3, multiple interleukins, GM-CSF, SCF, and CD30 by TAMs; and TNF-α and MIP-1α by Mo-MFs (all p < 0.05). Worse PTA was significantly correlated with higher secretion of IL-16 by VS cells (p < 0.05). Larger tumor size was significantly correlated with higher secretion of eotaxin by VS cells, and of IL-7, IL-21, and LIF by TAMs (all p = 0.017).Differential secretion of immune-related factors was observed in schwannoma versus normal SCs and in TAMs versus Mo-MFs, some of which were correlated with worse hearing and larger VS tumors.N/A Laryngoscope, 2023.

  View details for DOI 10.1002/lary.31067

  View details for PubMedID 37776249

• Potential Ototoxicity of Insulin-like Growth Factor 1 Receptor Signaling Inhibitors: An In Silico Drug Repurposing Study of the Regenerating Cochlear Neuron Transcriptome. Journal of clinical medicine Bertagnoli, L. E., Seist, R., Batts, S., Stankovic, K. M. 2023; 12 (10)

  Abstract

  Spiral ganglion neurons (SGNs) connect cochlear hair cells with higher auditory pathways and their degeneration due to drug toxicity (ototoxicity) contributes to hearing loss. This study aimed to identify drug classes that are negatively correlated with the transcriptome of regenerating SGNs. Human orthologs of differentially expressed genes within the regenerating neonatal mouse SGN transcriptome were entered into CMap and the LINCS unified environment and perturbation-driven gene expression was analyzed. The CMap connectivity scores ranged from 100 (positive correlation) to -100 (negative correlation). Insulin-like growth factor 1/receptor (IGF-1/R) inhibitors were highly negatively correlated with the regenerating SGN transcriptome (connectivity score: -98.87). A systematic literature review of clinical trials and observational studies reporting otologic adverse events (AEs) with IGF-1/R inhibitors identified 108 reports (6141 treated patients). Overall, 16.9% of the treated patients experienced any otologic AE; the rate was highest for teprotumumab (42.9%). In a meta-analysis of two randomized placebo-controlled trials of teprotumumab, there was a significantly higher risk of hearing-related (pooled Peto OR [95% CI]: 7.95 [1.57, 40.17]) and of any otologic AEs (3.56 [1.35, 9.43]) with teprotumumab vs. a placebo, whether or not dizziness/vertigo AEs were included. These results call for close audiological monitoring during IGF-1-targeted treatment, with prompt referral to an otolaryngologist should otologic AEs develop.

  View details for DOI 10.3390/jcm12103485

  View details for PubMedID 37240591

• The Future of Vestibular Schwannoma Management. Otolaryngologic clinics of North America Moore, L. S., Stankovic, K. M. 2023

  Abstract

  The future of the management of both sporadic and neurofibromatosis type 2-asscoiated vestibular schwannomas (VSs) will be shaped by cutting-edge technologic and biomedical advances to enable personalized, precision medicine. This scoping review envisions the future by highlighting the most promising developments published, ongoing, planned, or potential that are relevant for VS, including integrated omics approaches, artificial intelligence algorithms, biomarkers, liquid biopsy of the inner ear, digital medicine, inner ear endomicroscopy, targeted molecular imaging, patient-specific stem cell-derived models, ultra-high dose rate radiotherapy, optical imaging-guided microsurgery, high-throughput development of targeted therapeutics, novel immunotherapeutic strategies, tumor vaccines, and gene therapy.

  View details for DOI 10.1016/j.otc.2023.02.018

  View details for PubMedID 37019772

• Comparative Transcriptomic Analysis of Archival Human Vestibular Schwannoma Tissue from Patients with and without Tinnitus. Journal of clinical medicine Bommakanti, K., Seist, R., Kukutla, P., Cetinbas, M., Batts, S., Sadreyev, R. I., Stemmer-Rachamimov, A., Brenner, G. J., Stankovic, K. M. 2023; 12 (7)

  Abstract

  Vestibular schwannoma (VS) is an intracranial tumor that commonly presents with tinnitus and hearing loss. To uncover the molecular mechanisms underlying VS-associated tinnitus, we applied next-generation sequencing (Illumina HiSeq) to formalin-fixed paraffin-embedded archival VS samples from nine patients with tinnitus (VS-Tin) and seven patients without tinnitus (VS-NoTin). Bioinformatic analysis was used to detect differentially expressed genes (DEG; i.e., ≥two-fold change [FC]) while correcting for multiple comparisons. Using RNA-seq analysis, VS-Tin had significantly lower expression of GFAP (logFC = -3.04), APLNR (logFC = -2.95), PREX2 (logFC = -1.44), and PLVAP (logFC = -1.04; all p < 0.01) vs. VS-NoTin. These trends were validated by using real-time RT-qPCR. At the protein level, immunohistochemistry revealed a trend for less PREX2 and apelin expression and greater expression of NLRP3 inflammasome and CD68-positive macrophages in VS-Tin than in VS-NoTin, suggesting the activation of inflammatory processes in VS-Tin. Functional enrichment analysis revealed that the top three protein categories-glycoproteins, signal peptides, and secreted proteins-were significantly enriched in VS-Tin in comparison with VS-NoTin. In a gene set enrichment analysis, the top pathway was allograft rejection, an inflammatory pathway that includes the MMP9, CXCL9, IL16, PF4, ITK, and ACVR2A genes. Future studies are needed to examine the importance of these candidates and of inflammation in VS-associated tinnitus.

  View details for DOI 10.3390/jcm12072642

  View details for PubMedID 37048724

• Noise-Induced Hearing Loss. Journal of clinical medicine Natarajan, N., Batts, S., Stankovic, K. M. 2023; 12 (6)

  Abstract

  Noise-induced hearing loss (NIHL) is the second most common cause of sensorineural hearing loss, after age-related hearing loss, and affects approximately 5% of the world's population. NIHL is associated with substantial physical, mental, social, and economic impacts at the patient and societal levels. Stress and social isolation in patients' workplace and personal lives contribute to quality-of-life decrements which may often go undetected. The pathophysiology of NIHL is multifactorial and complex, encompassing genetic and environmental factors with substantial occupational contributions. The diagnosis and screening of NIHL are conducted by reviewing a patient's history of noise exposure, audiograms, speech-in-noise test results, and measurements of distortion product otoacoustic emissions and auditory brainstem response. Essential aspects of decreasing the burden of NIHL are prevention and early detection, such as implementation of educational and screening programs in routine primary care and specialty clinics. Additionally, current research on the pharmacological treatment of NIHL includes anti-inflammatory, antioxidant, anti-excitatory, and anti-apoptotic agents. Although there have been substantial advances in understanding the pathophysiology of NIHL, there remain low levels of evidence for effective pharmacotherapeutic interventions. Future directions should include personalized prevention and targeted treatment strategies based on a holistic view of an individual's occupation, genetics, and pathology.

  View details for DOI 10.3390/jcm12062347

  View details for PubMedID 36983347

• Association of Plasma Metabolomic Biomarkers With Persistent Tinnitus: A Population-Based Case-Control Study. JAMA otolaryngology-- head & neck surgery Zeleznik, O. A., Welling, D. B., Stankovic, K., Frueh, L., Balasubramanian, R., Curhan, G. C., Curhan, S. G. 2023

  Abstract

  Persistent tinnitus is common, disabling, and difficult to treat.To evaluate the association between circulating metabolites and persistent tinnitus.This was a population-based case-control study of 6477 women who were participants in the Nurses' Health Study (NHS) and NHS II with metabolomic profiles and tinnitus data. Information on tinnitus onset and frequency was collected on biennial questionnaires (2009-2017). For cases, metabolomic profiles were measured (2015-2021) in blood samples collected after the date of the participant's first report of persistent tinnitus (NHS, 1989-1999 and 2010-2012; NHS II, 1996-1999). Data analyses were performed from January 24, 2022, to January 14, 2023.In total, 466 plasma metabolites from 488 cases of persistent tinnitus and 5989 controls were profiled using 3 complementary liquid chromatography tandem mass spectrometry approaches.Logistic regression was used to estimate odds ratios (ORs) of persistent tinnitus (per 1 SD increase in metabolite values) and 95% CIs for each individual metabolite. Metabolite set enrichment analysis was used to identify metabolite classes enriched for associations with tinnitus.Of the 6477 study participants (mean [SD] age, 52 [9] years; 6477 [100%] female; 6121 [95%] White individuals) who were registered nurses, 488 reported experiencing daily persistent (≥5 minutes) tinnitus. Compared with participants with no tinnitus (5989 controls), those with persistent tinnitus were slightly older (53.0 vs 51.8 years) and more likely to be postmenopausal, using oral postmenopausal hormone therapy, and have type 2 diabetes, hypertension, and/or hearing loss at baseline. Compared with controls, homocitrulline (OR, 1.32; (95% CI, 1.16-1.50); C38:6 phosphatidylethanolamine (PE; OR, 1.24; 95% CIs, 1.12-1.38), C52:6 triglyceride (TAG; OR, 1.22; 95% CIs, 1.10-1.36), C36:4 PE (OR, 1.22; 95% CIs, 1.10-1.35), C40:6 PE (OR, 1.22; 95% CIs, 1.09-1.35), and C56:7 TAG (OR, 1.21; 95% CIs, 1.09-1.34) were positively associated, whereas α-keto-β-methylvalerate (OR, 0.68; 95% CIs, 0.56-0.82) and levulinate (OR, 0.60; 95% CIs, 0.46-0.79) were inversely associated with persistent tinnitus. Among metabolite classes, TAGs (normalized enrichment score [NES], 2.68), PEs (NES, 2.48), and diglycerides (NES, 1.65) were positively associated, whereas phosphatidylcholine plasmalogens (NES, -1.91), lysophosphatidylcholines (NES, -2.23), and cholesteryl esters (NES,-2.31) were inversely associated with persistent tinnitus.This population-based case-control study of metabolomic profiles and tinnitus identified novel plasma metabolites and metabolite classes that were significantly associated with persistent tinnitus, suggesting that metabolomic studies may help improve understanding of tinnitus pathophysiology and identify therapeutic targets for this challenging disorder.

  View details for DOI 10.1001/jamaoto.2023.0052

  View details for PubMedID 36928544

• Associations of Tinnitus Incidence with Use of Tumor Necrosis Factor-Alpha Inhibitors among Patients with Autoimmune Conditions. Journal of clinical medicine Natarajan, N., Batts, S., Gombar, S., Manickam, R., Sagi, V., Curhan, S. G., Stankovic, K. M. 2023; 12 (5)

  Abstract

  Tumor necrosis factor-alpha (TNFalpha) may promote neuroinflammation prompting tinnitus. This retrospective cohort study evaluated whether anti-TNFalpha therapy influences incident tinnitus risk among adults with autoimmune disorders and no baseline tinnitus selected from a US electronic health records database (Eversana; 1 January 2010-27 January 2022). Patients with anti-TNFalpha had ≥90-day history pre-index (first autoimmune disorder diagnosis) and ≥180-day follow-up post-index. Random samples (n = 25,000) of autoimmune patients without anti-TNFalpha were selected for comparisons. Tinnitus incidence was compared among patients with or without anti-TNFalpha therapy, overall and among at-risk age groups or by anti-TNFalpha category. High-dimensionality propensity score (hdPS) matching was used to adjust for baseline confounders. Compared with patients with no anti-TNFalpha, anti-TNFalpha was not associated with tinnitus risk overall (hdPS-matched HR [95% CI]: 1.06 [0.85, 1.33]), or between groups stratified by age (30-50 years: 1 [0.68, 1.48]; 51-70 years: 1.18 [0.89, 1.56]) or anti-TNFalpha category (monoclonal antibody vs. fusion protein: 0.91 [0.59, 1.41]). Anti-TNFalpha was not associated with tinnitus risk among those treated for ≥6 months (hdPS-matched HR [95% CI]: 0.96 [0.69, 1.32]) or ≥12 (1.03 [0.71, 1.5]), or those with RA (1.16 [0.88, 1.53]). Thus, in this US cohort study, anti-TNFalpha therapy was not associated with tinnitus incidence among patients with autoimmune disorders.

  View details for DOI 10.3390/jcm12051935

  View details for PubMedID 36902722

• Genome-wide screen of otosclerosis in population biobanks: 27 loci and shared associations with skeletal structure. Nature communications Ramo, J. T., Kiiskinen, T., Seist, R., Krebs, K., Kanai, M., Karjalainen, J., Kurki, M., Hamalainen, E., Happola, P., Havulinna, A. S., Hautakangas, H., FinnGen, Magi, R., Palta, P., Esko, T., Metspalu, A., Pirinen, M., Karczewski, K. J., Ripatti, S., Milani, L., Stankovic, K. M., Makitie, A., Daly, M. J., Palotie, A. 2023; 14 (1): 157

  Abstract

  Otosclerosis is one of the most common causes of conductive hearing loss, affecting 0.3% of the population. It typically presents in adulthood and half of the patients have a positive family history. The pathophysiology of otosclerosis is poorly understood. A previous genome-wide association study (GWAS) identified a single association locus in an intronic region of RELN. Here, we report a meta-analysis of GWAS studies of otosclerosis in three population-based biobanks comprising 3504 cases and 861,198 controls. We identify 23 novel risk loci (p<5*10-8) and report an association in RELN and three previously reported candidate gene or linkage regions (TGFB1, MEPE, and OTSC7). We demonstrate developmental stage-dependent immunostaining patterns of MEPE and RUNX2 in mouse otic capsules. In most association loci, the nearest protein-coding genes are implicated in bone remodelling, mineralization or severe skeletal disorders. We highlight multiple genes involved in transforming growth factor beta signalling for follow-up studies.

  View details for DOI 10.1038/s41467-022-32936-3

  View details for PubMedID 36653343

• Mortui vivos docent: a modern revival of temporal bone plug harvests. Frontiers in neuroscience Sagi, V., Kosaraju, N., Moore, L. S., Mulders, J. Y., Solyali, M., Ma, X., Regula, D. P., Hooper, J. E., Stankovic, K. M. 2023; 17: 1242831

  Abstract

  Human temporal bones (HTBs) are invaluable resources for the study of otologic disorders and for evaluating novel treatment approaches. Given the high costs and technical expertise required to collect and process HTBs, there has been a decline in the number of otopathology laboratories. Our objective is to encourage ongoing study of HTBs by outlining the necessary steps to establish a pipeline for collection and processing of HTBs. In this methods manuscript, we: (1) provide the design of a temporal bone plug sawblade that can be used to collect specimens from autopsy donors; (2) establish that decalcification time can be dramatically reduced from 9 to 3months if ethylenediaminetetraacetic acid is combined with microwave tissue processing and periodic bone trimming; (3) show that serial sections of relatively-rapidly decalcified HTBs can be successfully immunostained for key inner ear proteins; (4) demonstrate how to drill down a HTB to the otic capsule within a few hours so that subsequent decalcification time can be further reduced to only weeks. We include photographs and videos to facilitate rapid dissemination of the developed methods. Collected HTBs can be used for many purposes, including, but not limited to device testing, imaging studies, education, histopathology, and molecular studies. As new technology develops, it is imperative to continue studying HTBs to further our understanding of the cellular and molecular underpinnings of otologic disorders.

  View details for DOI 10.3389/fnins.2023.1242831

  View details for PubMedID 37886674

• miR-431 secreted by human vestibular schwannomas increases the mammalian inner ear's vulnerability to noise trauma. Frontiers in neurology Fujita, T., Seist, R., Kao, S., Soares, V., Panano, L., Khetani, R. S., Landegger, L. D., Batts, S., Stankovic, K. M. 2023; 14: 1268359

  Abstract

  Introduction: Vestibular schwannoma (VS) is an intracranial tumor that arises on the vestibular branch of cranial nerve VIII and typically presents with sensorineural hearing loss (SNHL). The mechanisms of this SNHL are postulated to involve alterations in the inner ear's microenvironment mediated by the genetic cargo of VS-secreted extracellular vesicles (EVs). We aimed to identify the EV cargo associated with poor hearing and determine whether its delivery caused hearing loss and cochlear damage in a mouse model in vivo.Methods: VS tissue was collected from routinely resected tumors of patients with good (VS-GH) or poor (VS-PH) pre-surgical hearing measured via pure-tone average and word recognition scores. Next-generation sequencing was performed on RNA isolated from cultured primary human VS cells and EVs from VS-conditioned media, stratified by patients' hearing ability. microRNA expression levels were compared between VS-PH and VS-GH samples to identify differentially expressed candidates for packaging into a synthetic adeno-associated viral vector (Anc80L65). Viral vectors containing candidate microRNA were infused to the semicircular canals of mice to evaluate the effects on hearing, including after noise exposure.Results: Differentially expressed microRNAs included hsa-miR-431-5p (enriched in VS-PH) and hsa-miR-192-5p (enriched in VS-GH). Newborn mice receiving intracochlear injection of viral vectors over-expressing hsa-miR-431-GFP, hsa-miR-192-GFP, or GFP only (control) had similar hearing 6weeks post-injection. However, after acoustic trauma, the miR-431 group displayed significantly worse hearing, and greater loss of synaptic ribbons per inner hair cell in the acoustically traumatized cochlear region than the control group.Conclusion: Our results suggest that miR-431 contributes to VS-associated hearing loss following cochlear stress. Further investigation is needed to determine whether miR-431 is a potential therapeutic target for SNHL.

  View details for DOI 10.3389/fneur.2023.1268359

  View details for PubMedID 37885485

• Polystyrene nanoplastics affect transcriptomic and epigenomic signatures of human fibroblasts and derived induced pluripotent stem cells: Implications for human health. Environmental pollution (Barking, Essex : 1987) Stojkovic, M., Ortuño Guzmán, F. M., Han, D., Stojkovic, P., Dopazo, J., Stankovic, K. M. 2022: 120849

  Abstract

  Plastic pollution is increasing at an alarming rate yet the impact of this pollution on human health is poorly understood. Because human induced pluripotent stem cells (hiPSC) are frequently derived from dermal fibroblasts, these cells offer a powerful platform for the identification of molecular biomarkers of environmental pollution in human cells. Here, we describe a novel proof-of-concept for deriving hiPSC from human dermal fibroblasts deliberately exposed to polystyrene (PS) nanoplastic particles; unexposed hiPSC served as controls. In parallel, unexposed hiPSC were exposed to low and high concentrations of PS nanoparticles. Transcriptomic and epigenomic signatures of all fibroblasts and hiPSCs were defined using RNA-seq and whole genome methyl-seq, respectively. Both PS-treated fibroblasts and derived hiPSC showed alterations in expression of ESRRB and HNF1A genes and circuits involved in the pluripotency of stem cells, as well as in pathways involved in cancer, inflammatory disorders, gluconeogenesis, carbohydrate metabolism, innate immunity, and dopaminergic synapse. Similarly, the expression levels of identified key transcriptional and DNA methylation changes (DNMT3A, ESSRB, FAM133CP, HNF1A, SEPTIN7P8, and TTC34) were significantly affected in both PS-exposed fibroblasts and hiPSC. This study illustrates the power of human cellular models of environmental pollution to narrow down and prioritize the list of candidate molecular biomarkers of environmental pollution. This knowledge will facilitate the deciphering of the origins of environmental diseases.

  View details for DOI 10.1016/j.envpol.2022.120849

  View details for PubMedID 36509347

• A fluorescent photoimmunoconjugate for imaging of cholesteatoma. Scientific reports Early, S., Saad, M. A., Mallidi, S., Mansour, A., Seist, R., Hasan, T., Stankovic, K. M. 2022; 12 (1): 19905

  Abstract

  Cholesteatoma is a potentially serious complication of chronic ear infections and requires surgical intervention for definitive management. Long-term complications include a frequent need for repeat surgical intervention for disease recurrence, and techniques to improve efficacy of single-stage surgery are an important area of continued research. This study investigates a novel application of the photosensitizer immune conjugate (PIC) cetuximab-benzoporphyrin derivative (Cet-BPD) for in vitro localization of human cholesteatoma tissue, coupled with an in vivo safety study for middle ear application of Cet-BPD in a murine model. In fresh human cholesteatoma tissues, Cet-BPD demonstrates selective localization to the hyperplastic squamous cell tissue associated with cholesteatoma, without localizing to other tissues such as middle ear mucosa. Applied to the murine middle ear, Cet-BPD does not demonstrate any deleterious effect on murine hearing when assessed by any of auditory brainstem response (ABR) thresholds, distortion product otoacoustic emission thresholds, or ABR wave I amplitudes. These findings demonstrate the technical promise and encouraging safety profile for the use of PICs for intraoperative localization and treatment of cholesteatoma.

  View details for DOI 10.1038/s41598-022-22072-9

  View details for PubMedID 36402793

• Toward Personalized Diagnosis and Therapy for Hearing Loss: Insights From Cochlear Implants. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology Sagi, V., Stankovic, K. M. 2022; 43 (8): e903-e909

  Abstract

  Sensorineural hearing loss (SNHL) is the most common sensory deficit, disabling nearly half a billion people worldwide. The cochlear implant (CI) has transformed the treatment of patients with SNHL, having restored hearing to more than 800,000 people. The success of CIs has inspired multidisciplinary efforts to address the unmet need for personalized, cellular-level diagnosis, and treatment of patients with SNHL. Current limitations include an inability to safely and accurately image at high resolution and biopsy the inner ear, precluding the use of key structural and molecular information during diagnostic and treatment decisions. Furthermore, there remains a lack of pharmacological therapies for hearing loss, which can partially be attributed to challenges associated with new drug development. We highlight advances in diagnostic and therapeutic strategies for SNHL that will help accelerate the push toward precision medicine. In addition, we discuss technological improvements for the CI that will further enhance its functionality for future patients. This report highlights work that was originally presented by Dr. Stankovic as part of the Dr. John Niparko Memorial Lecture during the 2021 American Cochlear Implant Alliance annual meeting.

  View details for DOI 10.1097/MAO.0000000000003624

  View details for PubMedID 35970169

• Association of Stapedotomy Volume and Patient Sex With Better Outcome. JAMA otolaryngology-- head & neck surgery Cahill, G., Wang, A. A., Lee, K., Sakagami, M., Welling, D. B., Stankovic, K. M. 2022

  View details for DOI 10.1001/jamaoto.2022.2142

  View details for PubMedID 35925579

• Magnetic stimulation allows focal activation of the mouse cochlea. eLife Lee, J. I., Seist, R., McInturff, S., Lee, D. J., Brown, M. C., Stankovic, K. M., Fried, S. 2022; 11

  Abstract

  Cochlear implants (CIs) provide sound and speech sensations for patients with severe to profound hearing loss by electrically stimulating the auditory nerve. While most CI users achieve some degree of open set word recognition under quiet conditions, hearing that utilizes complex neural coding (e.g., appreciating music) has proved elusive, probably because of the inability of CIs to create narrow regions of spectral activation. Several novel approaches have recently shown promise for improving spatial selectivity, but substantial design differences from conventional CIs will necessitate much additional safety and efficacy testing before clinical viability is established. Outside the cochlea, magnetic stimulation from small coils (micro-coils) has been shown to confine activation more narrowly than that from conventional microelectrodes, raising the possibility that coil-based stimulation of the cochlea could improve the spectral resolution of CIs. To explore this, we delivered magnetic stimulation from micro-coils to multiple locations of the cochlea and measured the spread of activation utilizing a multielectrode array inserted into the inferior colliculus; responses to magnetic stimulation were compared to analogous experiments with conventional microelectrodes as well as to responses when presenting auditory monotones. Encouragingly, the extent of activation with micro-coils was ~60% narrower compared to electric stimulation and largely similar to the spread arising from acoustic stimulation. The dynamic range of coils was more than three times larger than that of electrodes, further supporting a smaller spread of activation. While much additional testing is required, these results support the notion that magnetic micro-coil CIs can produce a larger number of independent spectral channels and may therefore improve auditory outcomes. Further, because coil-based devices are structurally similar to existing CIs, fewer impediments to clinical translational are likely to arise.

  View details for DOI 10.7554/eLife.76682

  View details for PubMedID 35608242

• Celastrol suppresses the growth of vestibular schwannoma in mice by promoting the degradation of beta-catenin. Acta pharmacologica Sinica Kim, N. H., Kwon, M., Jung, J., Chae, H. B., Lee, J., Yoon, Y., Moon, I. S., Lee, H. K., Namkung, W., Stankovic, K. M., Lee, S. A., Lee, J. D., Park, S. 2022

  Abstract

  Vestibular schwannoma (VS), one of characteristic tumors of neurofibromatosis type 2 (NF2), is an intracranial tumor that arises from Schwann cells of the vestibular nerve. VS results in hearing loss, tinnitus, dizziness, and even death, but there are currently no FDA-approved drugs for treatment. In this study, we established a high-throughput screening to discover effective compounds that could inhibit the viability of VS cells. Among 1019 natural products from the Korea Chemical Bank screened, we found that celastrol, a pentacyclic triterpene derived from a Tripterygium Wilfordi plant, exerted potent inhibitory effect on the viability of VS cells with an IC50 value of 0.5M. Celastrol (0.5, 1M) dose-dependently inhibited the proliferation of primary VS cells derived from VS patients. Celastrol also inhibited the growth, and induced apoptosis of two other VS cell lines (HEI-193 and SC4). Aberrant activation of Wnt/beta-catenin signaling has been found in VS isolated from clinically defined NF2 patients. In HEI-193 and SC4 cells, we demonstrated that celastrol (0.1, 0.5 muM) dose-dependently inhibited TOPFlash reporter activity and protein expression of beta-catenin, but not mRNA level of beta-catenin. Furthermore, celastrol accelerated the degradation of beta-catenin by promoting the formation of the beta-catenin destruction complex. In nude mice bearing VS cell line SC4 allografts, administration of celastrol (1.25mg·kg-1·d-1, i.p. once every 3 days for 2 weeks) significantly suppressed the tumor growth without showing toxicity. Collectively, this study demonstrates that celastrol can inhibit Wnt/beta-catenin signaling by promoting the degradation of beta-catenin, consequently inhibiting the growth of VS.

  View details for DOI 10.1038/s41401-022-00908-4

  View details for PubMedID 35478244

• Infectious Complications Following Cochlear Implant: Risk Factors, Natural History, and Management Patterns. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery Moon, P. K., Qian, Z. J., Ahmad, I. N., Stankovic, K. M., Chang, K. W., Cheng, A. G. 2022: 1945998221082530

  Abstract

  OBJECTIVE: To describe the natural history, detail the treatment patterns, and identify the risk factors for cochlear implant (CI) infections in a large US cohort.STUDY DESIGN: Retrospective study based on insurance claims.SETTING: Optum Data Mart database: 6101 patients who received CIs from 2003 to 2019.METHODS: Infections, treatments patterns, and timelines were described. A multivariable logistic regression model was used to assess the association between postoperative oral antibiotics and CI infection.RESULTS: The cohort includes 4736 (77.6%) adults and 1365 (22.4%) children. Between adult and pediatric patients, rates of CI infection (5.1% vs 4.5%, P = .18) and explantation (1.2% vs 0.8%, P = .11) were not significantly different. Infections typically occurred within 5 months of surgery. Children were diagnosed with CI infection earlier than adults (median difference, -1.5 months; P = .001). Postoperative oral antibiotic supply was not associated with lower risk of CI infection in either children or adults. However, among adults, otitis media was associated with higher odds of CI infection (odds ratio, 1.41; P < .001), while higher income was associated with lower odds of CI infection (odds ratio, 0.71; P = .03).CONCLUSIONS: Postoperative oral antibiotics were not associated with lower risk of infection or interventions. Otitis media episodes and lower income were associated with increased risk of infection among adults as well as intervention overall. Infection typically presented within the first 6 months after surgery, with children presenting earlier than adults. Overall, our findings serve as a resource for providers to consider in their care of patients with CIs.

  View details for DOI 10.1177/01945998221082530

  View details for PubMedID 35192408

• Human vestibular schwannoma reduces density of auditory nerve fibers in the osseous spiral lamina. Hearing research Eggink, M. C., Frijns, J. H., Sagers, J. E., O'Malley, J. T., Liberman, M. C., Stankovic, K. M. 2022; 418: 108458

  Abstract

  Hearing loss in patients with vestibular schwannoma (VS) is commonly attributed to mechanical compression of the auditory nerve, though recent studies suggest that this retrocochlear pathology may be augmented by cochlear damage. Although VS-associated loss of inner hair cells, outer hair cells, and spiral ganglion cells has been reported, it is unclear to what extent auditory-nerve peripheral axons are damaged in VS patients. Understanding the degree of damage VSs cause to auditory nerve fibers (ANFs) is important for accurately modeling clinical outcomes of cochlear implantation, which is a therapeutic option to rehabilitate hearing in VS-affected ears. A retrospective analysis of human temporal-bone histopathology was performed on archival specimens from the Massachusetts Eye and Ear collection. Seven patients met our inclusion criteria based on the presence of sporadic, unilateral, untreated VS. Tangential sections of five cochlear regions were stained with hematoxylin and eosin, and adjacent sections were stained to visualize myelinated ANFs and efferent fibers. Following confocal microscopy, peripheral axons of ANFs within the osseous spiral lamina were quantified manually, where feasible, and with a "pixel counting" method, applicable to all sections. ANF density was substantially reduced on the VS side compared to the unaffected contralateral side. In the upper basal turn, a significant difference between the VS side and unaffected contralateral side was found using both counting methods, corresponding to the region tuned to 2000Hz. Even spiral ganglion cells (SGCs) contralateral to VS were affected by the tumor as the majority of contralateral SGC counts were below average for age. This observation provides histological insight into the clinical observation that unilateral vestibular schwannomas pose a long-term risk of progression of hearing loss in the contralateral ear as well. Our pixel counting method for ANF quantification in the osseous spiral lamina is applicable to other pathologies involving sensorineural hearing loss. Future research is needed to classify ANFs into morphological categories, accurately predict their electrical properties, and use this knowledge to inform optimal cochlear implant programming strategies.

  View details for DOI 10.1016/j.heares.2022.108458

  View details for PubMedID 35334332

• Sporadic Vestibular Schwannoma Size and Location Do not Correlate With the Severity of Hearing Loss at Initial Presentation. Frontiers in oncology Brown, A., Early, S., Vasilijic, S., Stankovic, K. M. 2022; 12: 836504

  Abstract

  Vestibular schwannoma (VS) is a non-malignant intracranial neoplasm arising from the vestibular branch of the 8th cranial nerve; sensorineural hearing loss (SNHL) is the most common associated symptom. Understanding whether VS imaging characteristics at the time of VS diagnosis can be associated with severity of VS-induced SNHL can impact patient counseling and define promising areas for future research. Patients diagnosed with VS at Massachusetts Eye and Ear (MEE) from 1994 through 2018 were analyzed if magnetic resonance imaging at VS presentation and sequential audiometry were available. Results were compared with original studies available in PubMed, written in English, on VS imaging characteristics and their impact on hearing in patients. A total of 477 patients with unilateral VS from the MEE database demonstrated no significant correlation between any features of tumor imaging at the time of VS diagnosis, such as VS size, impaction or location, and any hearing loss metric. Twenty-three published studies on the impact of VS imaging characteristics on patient hearing met inclusion criteria, with six solely involving NF2 patients and three including both sporadic and NF2-related VS patients. Fifteen studies reported a significant relationship between SNHL and at least one VS imaging characteristic; however, these trends were universally limited to NF2 patients or involved small patient populations, and were not reproduced in larger studies. Taken together, SNHL in sporadic VS patients is not readily associated solely with any tumor imaging characteristics. This finding motivates future studies to define how VS microenvironment and secreted molecules influence VS-induced SNHL.

  View details for DOI 10.3389/fonc.2022.836504

  View details for PubMedID 35372070

• Choice of vector and surgical approach enables efficient cochlear gene transfer in nonhuman primate. Nature communications Andres-Mateos, E., Landegger, L. D., Unzu, C., Phillips, J., Lin, B. M., Dewyer, N. A., Sanmiguel, J., Nicolaou, F., Valero, M. D., Bourdeu, K. I., Sewell, W. F., Beiler, R. J., McKenna, M. J., Stankovic, K. M., Vandenberghe, L. H. 2022; 13 (1): 1359

  Abstract

  Inner ear gene therapy using adeno-associated viral vectors (AAV) promises to alleviate hearing and balance disorders. We previously established the benefits of Anc80L65 in targeting inner and outer hair cells in newborn mice. To accelerate translation to humans, we now report the feasibility and efficiency of the surgical approach and vector delivery in a nonhuman primate model. Five rhesus macaques were injected with AAV1 or Anc80L65 expressing eGFP using a transmastoid posterior tympanotomy approach to access the round window membrane after making a small fenestra in the oval window. The procedure was well tolerated. All but one animal showed cochlear eGFP expression 7-14 days following injection. Anc80L65 in 2 animals transduced up to 90% of apical inner hair cells; AAV1 was markedly less efficient at equal dose. Transduction for both vectors declined from apex to base. These data motivate future translational studies to evaluate gene therapy for human hearing disorders.

  View details for DOI 10.1038/s41467-022-28969-3

  View details for PubMedID 35292639

• Reversible contrast enhancement for visualization of human temporal bones using micro computed tomography. Frontiers in surgery Bommakanti, K. K., Iyer, J. S., Sagi, V., Brown, A., Ma, X., Gonzales, M., Stankovic, K. M. 2022; 9: 952348

  Abstract

  Sensorineural hearing loss (SNHL), which typically arises from the inner ear, is the most common sensory deficit worldwide. The traditional method for studying pathophysiology underlying human SNHL involves histological processing of the inner ear from temporal bones collected during autopsy. Histopathological analysis is destructive and limits future use of a given specimen. Non-destructive strategies for the study of the inner ear are urgently needed to fully leverage the utility of each specimen because access to human temporal bones is increasingly difficult and these precious specimens are required to uncover disease mechanisms and to enable development of new devices. We highlight the potential of reversible iodine staining for micro-computed tomography imaging of the human inner ear. This approach provides reversible, high-resolution visualization of intracochlear structures and is becoming more rapid and accessible.

  View details for DOI 10.3389/fsurg.2022.952348

  View details for PubMedID 36268215

• Imbalance and dizziness caused by unilateral vestibular schwannomas correlate with vestibulo-ocular reflex precision and bias J Neurophysiol . King, S., Dahlem , K., Karmali , F., Stankovic, K. M., Welling, D. B., Lewis, R. F. 2022; 127 (2): 596-606

  View details for DOI 10.1152/jn.00725.2020

• Accelerated Long-Term Hearing Loss Progression After Recovery From Idiopathic Sudden Sensorineural Hearing Loss FRONTIERS IN NEUROLOGY Early, S., van der Valk, J. C., Frijns, J. M., Stankovic, K. M. 2021; 12: 738942

  Abstract

  Background and Introduction: Idiopathic sudden sensorineural hearing loss (ISSNHL) is characterized by rapid onset, typically unilateral presentation, and variable recovery. This case-control observational study aimed to improve patient counseling by objectively characterizing long-term hearing loss progression following ISSNHL, using sequential audiometry in the largest-to-date cohort of patients with ISSNHL. Methods: Patients diagnosed with ISSNHL at a tertiary referral hospital from 1994 through 2018 with sequential audiometry were studied. Case controls with sensorineural hearing loss (SNHL) were matched by age, sex, baseline hearing status, and frequency of sequential audiometry. Hearing loss progression was quantified using Kaplan-Meier (K-M) analysis to account for variable follow-up duration. A subgroup analysis was performed by age, sex, preexisting comorbidities, ISSNHL-associated symptoms, ISSNHL treatment, and degree of post-ISSNHL hearing recovery. Results: A total of 660 patients were identified with ISSNHL. In patients with post-ISSNHL recovery to good hearing [pure tone average (PTA) <30 dB and word recognition score (WRS) > 70%], median time to progression to non-serviceable (PTA > 50 dB or WRS <50%) SNHL was 16.4 years. In patients with incomplete post-ISSNHL hearing recovery, contralateral ears were also at significantly higher risk of SNHL progression over the following 12-year period. Male sex was associated with increased risk of SNHL progression [odds ratio (OR) 3.45 male vs. female] at 5-year follow up. No other subgroup factors influenced the likelihood of SNHL progression. Discussion and Conclusion: Patients should be counseled on continued risk to long-term hearing after stabilization of hearing post-ISSNHL, with particular emphasis on greater risk to the contralateral ear in those with incomplete ipsilateral recovery.

  View details for DOI 10.3389/fneur.2021.738942

  View details for Web of Science ID 000732825200001

  View details for PubMedID 34956043

  View details for PubMedCentralID PMC8693444

• Editorial: Emerging Ototoxic Medications and Their Role in Cochlear and Vestibular Disorders. Frontiers in neurology Szczepek, A. J., Stankovic, K. M. 2021; 12: 773714

  View details for DOI 10.3389/fneur.2021.773714

  View details for PubMedID 34744994

  View details for PubMedCentralID PMC8569918

• Implementation of Mobile Audiometry During the COVID-19 Pandemic OTOLARYNGOLOGY-HEAD AND NECK SURGERY Garcia, A., Chari, D. A., Stankovic, K. M., Lee, D. J., Kozin, E. D., Franck, K. H. 2021: 1945998211051588

  Abstract

  During the COVID-19 pandemic, the utility of portable audiometry became more apparent as elective procedures were deferred in an effort to limit exposure to health care providers. Herein, we retrospectively evaluated mobile-based audiometry in the emergency department and outpatient otology and audiology clinics. Air conduction thresholds with mobile audiometry were within 5 dB in 66% of tests (95% CI, 62.8%-69.09%) and within 10 dB in 84% of tests (95% CI, 81.4%-86.2%) as compared with conventional audiometry. No significant differences were noted between mobile-based and conventional audiometry at any frequencies, except 8 kHz (P < .05). The sensitivity and specificity for screening for hearing loss were 94.3% (95% CI, 91.9%-96.83%) and 92.3% (95% CI, 90.1%-94.4%), respectively. While automated threshold audiometry does not replace conventional audiometry, mobile audiometry is a promising screening tool when conventional audiometry is not available.

  View details for DOI 10.1177/01945998211051588

  View details for Web of Science ID 000705159600001

  View details for PubMedID 34609938

• Conductive hearing loss in the Hyp mouse model of X-linked hypophosphatemia is accompanied by hypomineralization of the auditory ossicles. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research Delsmann, M. M., Seist, R., Sturznickel, J., Schmidt, F. N., Mansour, A., Kobelski, M. M., Broocks, G., Peichl, J., Oheim, R., Praetorius, M., Schinke, T., Amling, M., Demay, M. B., Stankovic, K. M., Rolvien, T. 2021

  Abstract

  X-linked hypophosphatemia (XLH) is a hereditary musculoskeletal disorder caused by loss-of-function mutations in the PHEX gene. In XLH, increased circulating fibroblast growth factor 23 (FGF23) levels cause renal phosphate wasting and low concentrations of 1,25-dihydroxyvitamin D, leading to an early clinical manifestation of rickets. Importantly, hearing loss is commonly observed in XLH patients. We here present data from two XLH patients with marked conductive hearing loss. To decipher the underlying pathophysiology of hearing loss in XLH, we utilized the Hyp mouse model of XLH and measured auditory brain stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) to functionally assess hearing. As evidenced by the increased ABR/DPOAE threshold shifts in the mid-frequency range, these measurements indicated a predominantly conductive hearing loss in Hyp mice compared to wild type (WT) mice. Therefore, we carried out an in-depth histomorphometric and scanning electron microscopic analysis of the auditory ossicles. Quantitative backscattered electron imaging (qBEI) indicated a severe hypomineralization of the ossicles in Hyp mice, evidenced by lower calcium content (CaMean) and higher void volume (i.e., porosity) compared to WT mice. Histologically, voids correlated with unmineralized bone (i.e., osteoid), and the osteoid volume per bone volume (OV/BV) was markedly higher in Hyp mice than WT mice. The density of osteocyte lacunae was lower in Hyp mice than in WT mice, whereas osteocyte lacunae were enlarged. Taken together, our findings highlight the importance of ossicular mineralization for hearing conduction and point towards the potential benefit of improving mineralization to prevent hearing loss in XLH. This article is protected by copyright. All rights reserved.

  View details for DOI 10.1002/jbmr.4443

  View details for PubMedID 34523743

• Endomicroscopy of the human cochlea using a micro-optical coherence tomography catheter. Scientific reports Iyer, J. S., Yin, B., Stankovic, K. M., Tearney, G. J. 2021; 11 (1): 17932

  Abstract

  Sensorineural hearing loss (SNHL) is one of the most profound public health concerns of the modern era, affecting 466 million people today, and projected to affect 900 million by the year 2050. Advances in both diagnostics and therapeutics for SNHL have been impeded by the human cochlea's inaccessibility for in vivo imaging, resulting from its extremely small size, convoluted coiled configuration, fragility, and deep encasement in dense bone. Here, we develop and demonstrate the ability of a sub-millimeter-diameter, flexible endoscopic probe interfaced with a micro-optical coherence tomography (muOCT) imaging system to enable micron-scale imaging of the inner ear's sensory epithelium in cadaveric human inner ears.

  View details for DOI 10.1038/s41598-021-95991-8

  View details for PubMedID 34504113

• The AAV9 Variant Capsid AAV-F Mediates Widespread Transgene Expression in Nonhuman Primate Spinal Cord After Intrathecal Administration. Human gene therapy Beharry, A., Gong, Y., Kim, J. C., Hanlon, K. S., Nammour, J., Hieber, K., Eichler, F., Cheng, M., Stemmer-Rachamimov, A., Stankovic, K. M., Welling, D. B., Ng, C., Maguire, C. A. 2021

  Abstract

  Intrathecal delivery of AAV9 into the subarachnoid space has been shown to transduce spinal cord and brain and be less affected by preexisting antibodies, which are lower in cerebral spinal fluid. Still, efficiency of transduction needs to be improved. Recently, we identified a new capsid from a library selection in mice, called AAV-F, that allowed robust transduction of the spinal cord gray matter after lumbar injection. In this study, we test transduction of spinal cord by AAV-F (n = 3) compared to AAV9 (n = 2), using a reporter gene, in cynomolgus monkeys after lumbar intrathecal injection. Using an automated image analysis (IA) approach to sensitively quantitate reporter gene expression in spinal cord, we found that AAV-F capsid mediated slightly higher transgene expression (both in percentages of cells and intensity of immunostaining) in motor neurons and interneurons, in the lumbar and thoracic regions, compared to AAV9. Interestingly, although AAV-F mediated higher transgene expression in spinal cord, the number of genomes in spinal cord and periphery were on average lower for AAV-F than AAV9, which suggest that lower numbers of genomes were able to mediate higher transgene expression in spinal cord with this capsid. In contrast, dorsal root ganglion transduction efficiency was lower for AAV-F compared to AAV9 on average. Interestingly, we also observed transduction of Schwann cells in sciatic nerve in two nonhuman primates injected with AAV-F, but none with AAV9. Overall, our data demonstrate the utility of automated IA for quantitation of AAV transduction in the spinal cord and the favorable on-target:off-target transduction profile suggests that the AAV-F capsid be considered for gene therapy applications focused on treating the spinal cord after intrathecal delivery.

  View details for DOI 10.1089/hum.2021.069

  View details for PubMedID 34128391

• Re-engineering the Surgeon-Scientist Pipeline: Advancing Diversity and Equity to Fuel Scientific Innovation. The Laryngoscope Munjal, T., Nathan, C., Brenner, M. J., Stankovic, K. M., Francis, H. W., Valdez, T. A. 2021

  View details for DOI 10.1002/lary.29800

  View details for PubMedID 34383298

• Two Photon Fluorescence Microscopy of the Unstained Human Cochlea Reveals Organ of Corti Cytoarchitecture FRONTIERS IN CELLULAR NEUROSCIENCE Iyer, J. S., Seist, R., Moon, I., Stankovic, K. M. 2021; 15: 690953

  Abstract

  Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide, and it typically originates from the cochlea. Methods to visualize intracochlear cells in living people are currently lacking, limiting not only diagnostics but also therapies for SNHL. Two-photon fluorescence microscopy (TPFM) is a high-resolution optical imaging technique. Here we demonstrate that TPFM enables visualization of sensory cells and auditory nerve fibers in an unstained, non-decalcified adult human cochlea.

  View details for DOI 10.3389/fncel.2021.690953

  View details for Web of Science ID 000684747300004

  View details for PubMedID 34421541

  View details for PubMedCentralID PMC8376148

• Losartan prevents tumor-induced hearing loss and augments radiation efficacy in NF2 schwannoma rodent models. Science translational medicine Wu, L., Vasilijic, S., Sun, Y., Chen, J., Landegger, L. D., Zhang, Y., Zhou, W., Ren, J., Early, S., Yin, Z., Ho, W. W., Zhang, N., Gao, X., Lee, G. Y., Datta, M., Sagers, J. E., Brown, A., Muzikansky, A., Stemmer-Rachamimov, A., Zhang, L., Plotkin, S. R., Jain, R. K., Stankovic, K. M., Xu, L. 2021; 13 (602)

  Abstract

  Hearing loss is one of the most common symptoms of neurofibromatosis type 2 (NF2) caused by vestibular schwannomas (VSs). Fibrosis in the VS tumor microenvironment (TME) is associated with hearing loss in patients with NF2. We hypothesized that reducing the fibrosis using losartan, an FDA-approved antihypertensive drug that blocks fibrotic and inflammatory signaling, could improve hearing. Using NF2 mouse models, we found that losartan treatment normalized the TME by (i) reducing neuroinflammatory IL-6/STAT3 signaling and preventing hearing loss, (ii) normalizing tumor vasculature and alleviating neuro-edema, and (iii) increasing oxygen delivery and enhancing efficacy of radiation therapy. In preparation to translate these exciting findings into the clinic, we used patient samples and data and demonstrated that IL-6/STAT3 signaling inversely associated with hearing function, that elevated production of tumor-derived IL-6 was associated with reduced viability of cochlear sensory cells and neurons in ex vivo organotypic cochlear cultures, and that patients receiving angiotensin receptor blockers have no progression in VS-induced hearing loss compared with patients on other or no antihypertensives based on a retrospective analysis of patients with VS and hypertension. Our study provides the rationale and critical data for a prospective clinical trial of losartan in patients with VS.

  View details for DOI 10.1126/scitranslmed.abd4816

  View details for PubMedID 34261799

• Special Series: Stem Cells and Hearing Loss STEM CELLS Lako, M., Stankovic, K. M., Stojkovic, M. 2021; 39 (7): 835-837

  View details for DOI 10.1002/stem.3419

  View details for Web of Science ID 000667060300001

  View details for PubMedID 33951256

• Human induced pluripotent stem cells and CRISPR/Cas-mediated targeted genome editing: Platforms to tackle sensorineural hearing loss. Stem cells (Dayton, Ohio) Stojkovic, M., Han, D., Jeong, M., Stojkovic, P., Stankovic, K. M. 2021; 39 (6): 673-696

  Abstract

  Hearing loss (HL) is a major global health problem of pandemic proportions. The most common type of HL is sensorineural hearing loss (SNHL) which typically occurs when cells within the inner ear are damaged. Human induced pluripotent stem cells (hiPSCs) can be generated from any individual including those who suffer from different types of HL. The development of new differentiation protocols to obtain cells of the inner ear including hair cells (HCs) and spiral ganglion neurons (SGNs) promises to expedite cell-based therapy and screening of potential pharmacologic and genetic therapies using human models. Considering age-related, acoustic, ototoxic, and genetic insults which are the most frequent causes of irreversible damage of HCs and SGNs, new methods of genome editing (GE), especially the CRISPR/Cas9 technology, could bring additional opportunities to understand the pathogenesis of human SNHL and identify novel therapies. However, important challenges associated with both hiPSCs and GE need to be overcome before scientific discoveries are correctly translated to effective and patient-safe applications. The purpose of the present review is (a) to summarize the findings from published reports utilizing hiPSCs for studies of SNHL, hence complementing recent reviews focused on animal studies, and (b) to outline promising future directions for deciphering SNHL using disruptive molecular and genomic technologies.

  View details for DOI 10.1002/stem.3353

  View details for PubMedID 33586253

• Cochlin Deficiency Protects Against Noise-Induced Hearing Loss FRONTIERS IN MOLECULAR NEUROSCIENCE Seist, R., Landegger, L. D., Robertson, N. G., Vasilijic, S., Morton, C. C., Stankovic, K. M. 2021; 14: 670013

  Abstract

  Cochlin is the most abundant protein in the inner ear. To study its function in response to noise trauma, we exposed adolescent wild-type (Coch +/+ ) and cochlin knock-out (Coch -/-) mice to noise (8-16 kHz, 103 dB SPL, 2 h) that causes a permanent threshold shift and hair cell loss. Two weeks after noise exposure, Coch-/- mice had substantially less elevation in noise-induced auditory thresholds and hair cell loss than Coch + / + mice, consistent with cochlin deficiency providing protection from noise trauma. Comparison of pre-noise exposure thresholds of auditory brain stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) in Coch-/- mice and Coch + / + littermates revealed a small and significant elevation in thresholds of Coch-/- mice, overall consistent with a small conductive hearing loss in Coch-/- mice. We show quantitatively that the pro-inflammatory component of cochlin, LCCL, is upregulated after noise exposure in perilymph of wild-type mice compared to unexposed mice, as is the enzyme catalyzing LCCL release, aggrecanase1, encoded by Adamts4. We further show that upregulation of pro-inflammatory cytokines in perilymph and cochlear soft-tissue after noise exposure is lower in cochlin knock-out than wild-type mice. Taken together, our data demonstrate for the first time that cochlin deficiency results in conductive hearing loss that protects against physiologic and molecular effects of noise trauma.

  View details for DOI 10.3389/fnmol.2021.670013

  View details for Web of Science ID 000658161600001

  View details for PubMedID 34108864

  View details for PubMedCentralID PMC8180578

• Osteoporosis, bisphosphonate use, and risk of moderate or worse hearing loss in women JOURNAL OF THE AMERICAN GERIATRICS SOCIETY Curhan, S. G., Stankovic, K., Halpin, C., Wang, M., Eavey, R. D., Paik, J. M., Curhan, G. C. 2021

  Abstract

  Osteoporosis and low bone density (LBD) may be associated with higher risk of hearing loss, but findings are inconsistent and longitudinal data are scarce. Bisphosphonates may influence risk, but the relation has not been studied in humans. We longitudinally investigated associations of osteoporosis and LBD, bisphosphonate use, vertebral fracture (VF), hip fracture (HF), and risk of self-reported moderate or worse hearing loss.Longitudinal cohort study.The Nurses' Health Study (NHS) (1982-2016) and Nurses' Health Study II (NHS II) (1995-2017).Participants included 60,821 NHS women, aged 36-61 years at baseline, and 83,078 NHS II women, aged 31-48 years at baseline (total n = 143,899).Information on osteoporosis, LBD, bisphosphonate use, VF, HF, and hearing status was obtained from validated biennial questionnaires. In a subcohort (n = 3749), objective hearing thresholds were obtained by audiometry. Multivariable-adjusted Cox proportional hazards models were used to examine independent associations between osteoporosis (NHS), osteoporosis/LBD (NHS II), and risk of hearing loss.The multivariable-adjusted relative risk (MVRR, 95% confidence interval) of moderate or worse hearing loss was higher among women with osteoporosis or LBD in both cohorts. In NHS, compared with women without osteoporosis, the MVRR was 1.14 (1.09, 1.19) among women with osteoporosis; in NHS II, the MVRR was 1.30 (1.21, 1.40) among women with osteoporosis/LBD. The magnitude of the elevated risk was similar among women who did and did not use bisphosphonates. VF was associated with higher risk (NHS: 1.31 [1.16, 1.49]; NHS II: 1.39 [1.13, 1.71]), but HF was not (NHS: 1.00 [0.86, 1.16];NHS II: 1.15 [0.75,1.74]). Among participants with audiometric measurements, compared with women without osteoporosis/LBD, the mean multivariable-adjusted hearing thresholds were higher (i.e., worse) among those with osteoporosis/LBD who used bisphosphonates.Osteoporosis and LBD may be important contributors to aging-related hearing loss. Among women with osteoporosis, the risk of hearing loss was not influenced by bisphosphonate use.

  View details for DOI 10.1111/jgs.17275

  View details for Web of Science ID 000653121800001

  View details for PubMedID 34028002

• Postnatal expression and possible function of RANK and RANKL in the murine inner ear BONE Kao, S., Katsumi, S., Han, D., Bizaki-Vallaskangas, A. J., Vasilijic, S., Landegger, L. D., Kristiansen, A. G., McKenna, M. J., Stankovic, K. M. 2021; 145: 115837

  Abstract

  The bone encasing the inner ear, known as the otic capsule, is unique because it remodels little postnatally compared to other bones in the body. Previous studies established that osteoprotegerin (OPG) in the inner ear inhibits otic capsule remodeling. OPG acts as a decoy receptor of receptor activator of nuclear factor κB ligand (RANKL) to disrupt the interaction between RANKL and RANK, the primary regulators of bone metabolism. Here we studied the expression and function of RANK and RANKL in the murine cochlea. Using a combination of in situ hybridization, real-time quantitative RT-PCR, and western blot, we demonstrate that Rankl and Rank genes and their protein products are expressed in the intracochlear soft tissues and the otic capsule in a developmentally regulated manner. Using a culture of neonatal murine cochlear neurons, we show that the interaction between RANK and RANKL inhibits neurite outgrowth in these neurons, and is associated with upregulation of NOGO-A expression. Taken together, our results suggest that, in addition to regulating otic capsule bone remodeling, RANK and RANKL expressed by intracochlear soft tissues may also regulate spiral ganglion neuron function by affecting neurite outgrowth.

  View details for DOI 10.1016/j.bone.2020.115837

  View details for Web of Science ID 000623120700009

  View details for PubMedID 33385614

• Initial Method for Characterization of Tympanic Membrane Drug Permeability in Human Temporal Bones In Situ FRONTIERS IN NEUROLOGY Early, S., Yang, R., Li, X., Zhang, Z., van der Valk, J. C., Ma, X., Kohane, D. S., Stankovic, K. M. 2021; 12: 580392

  Abstract

  Background and Introduction: Acute otitis media is the most common reason for a visit to the pediatrician, often requiring systemic administration of oral antibiotics. Local drug therapy applied to the middle ear could avoid side effects associated with systemic antibiotic administration, however in the majority of patients this would require drugs to diffuse across an intact tympanic membrane. Experimental methods for testing trans-tympanic drug flux in human tissues in situ would be highly valuable to guide drug therapy development for local drug delivery to the middle ear. Materials and Methods: A total of 30 cadaveric human temporal bones were characterized by trans-tympanic impedance testing to determine how steps in tissue processing and storage might impact intactness of the tympanic membrane and thus suitability for use in studies of trans-tympanic drug flux. Ciprofloxacin drug solutions of varying concentrations were then applied to the lateral surface of the tympanic membrane in eight samples, and middle ear aspirate was collected over the following 48 h to evaluate trans-tympanic flux to the middle ear. Results: Tissue processing steps that involved extensive tissue manipulation were consistently associated with evidence of microperforations in the tympanic membrane tissue. Maintaining the tympanic membrane in situ within the temporal bone, while using an otologic drill to obtain transmastoid access to the middle ear, was demonstrated as a reliable, non-damaging technique for accessing both lateral and medial surfaces for trans-tympanic flux testing. Results in these bones demonstrated trans-tympanic flux of ciprofloxacin when administered at sufficiently high concentration. Discussion and Conclusion: The study describes key techniques and best practices, as well as pitfalls to avoid, in the development of a model for studying trans-tympanic drug flux in human temporal bones in situ. This model can be a valuable research tool in advancing progress toward eventual clinical studies for trans-tympanic drug delivery to the middle ear.

  View details for DOI 10.3389/fneur.2021.580392

  View details for Web of Science ID 000626459700001

  View details for PubMedID 33708167

  View details for PubMedCentralID PMC7940379

• Human pluripotent stem cells - Unique tools to decipher the effects of environmental and intracellular plastic pollution on human health. Environmental pollution (Barking, Essex : 1987) Stojkovic, M., Stojkovic, P., Stankovic, K. M. 2021; 269: 116144

  Abstract

  Increase in plastic pollution causes irreparable harm to the environment lasting for decades. While current data of plastic pollution include marine and terrestrial ecology, the impacts of degraded or intentionally produced microscopic-sized plastics on human health remain unknown. Here, we are proposing the usage of pluripotent stem cells, modern transcriptomics, and bioinformatics as a unique scientific tool to define the link between environmental and intracellular pollution, its outcome on early human development and origin of diseases. This commentary is an urgent appeal to the scientific and policy communities to invest more time and resources to establish reliable standards and methods to define and address the consequences of plastic pollution on human health.

  View details for DOI 10.1016/j.envpol.2020.116144

  View details for PubMedID 33288300

• Direct SARS-CoV-2 infection of the human inner ear may underlie COVID-19-associated audiovestibular dysfunction Communications Medicine Jeong, M. 2021; 1 (1)

  View details for DOI 10.1038/s43856-021-00044-w

• Molecular and Clinical Significance of Fibroblast Growth Factor 2 in Development and Regeneration of the Auditory System. Frontiers in molecular neuroscience Jeong, M., Bojkovic, K., Sagi, V., Stankovic, K. M. 1800; 14: 757441

  Abstract

  The fibroblast growth factor 2 (FGF2) is a member of the FGF family which is involved in key biological processes including development, cellular proliferation, wound healing, and angiogenesis. Although the utility of the FGF family as therapeutic agents has attracted attention, and FGF2 has been studied in several clinical contexts, there remains an incomplete understanding of the molecular and clinical function of FGF2 in the auditory system. In this review, we highlight the role of FGF2 in inner ear development and hearing protection and present relevant clinical studies for tympanic membrane (TM) repair. We conclude by discussing the future implications of FGF2 as a potential therapeutic agent.

  View details for DOI 10.3389/fnmol.2021.757441

  View details for PubMedID 35002617

• TMPRSS3 Gene Variants With Implications for Auditory Treatment and Counseling. Frontiers in genetics Moon, I. S., Grant, A. R., Sagi, V., Rehm, H. L., Stankovic, K. M. 2021; 12: 780874

  Abstract

  Objective: To identify and report novel variants in the TMPRSS3 gene and their clinical manifestations related to hearing loss as well as intervention outcomes. This information will be helpful for genetic counseling and treatment planning for these patients. Methods: Literature review of previously reported TMPRSS3 variants was conducted. Reported variants and associated clinical information was compiled. Additionally, cohort data from 18 patients, and their families, with a positive result for TMPRSS3-associated hearing loss were analyzed. Genetic testing included sequencing and copy number variation (CNV) analysis of TMPRSS3 and the Laboratory for Molecular Medicine's OtoGenome-v1, -v2, or -v3 panels. Clinical data regarding patient hearing rehabilitation was interpreted along with their genetic testing results and in the context of previously reported cochlear implant outcomes in individuals with TMPRSS3 variants. Results: There have been 87 previously reported TMPRSS3 variants associated with non-syndromic hearing loss in more than 20 ancestral groups worldwide. Here we report occurrences of known variants as well as one novel variant: deletion of Exons 1-5 and 13 identified from our cohort of 18 patients. The hearing impairment in many of these families was consistent with that of previously reported patients with TMPRSS3 variants (i.e., typical down-sloping audiogram). Four patients from our cohort underwent cochlear implantation. Conclusion: Bi-allelic variants of TMPRSS3 are associated with down-sloping hearing loss regardless of ancestry. The outcome following cochlear implantation in patients with variants of TMPRSS3 is excellent. Therefore, cochlear implantation is strongly recommended for hearing rehabilitation in these patients.

  View details for DOI 10.3389/fgene.2021.780874

  View details for PubMedID 34868270

• New developments in neurofibromatosis type 2 and vestibular schwannoma. Neuro-oncology advances Ren, Y., Chari, D. A., Vasilijic, S., Welling, D. B., Stankovic, K. M. 2020; 3 (1): vdaa153

  Abstract

  Neurofibromatosis type 2 (NF2) is a rare autosomal dominant disorder characterized by the development of multiple nervous system tumors due to mutation in the NF2 tumor suppressor gene. The hallmark feature of the NF2 syndrome is the development of bilateral vestibular schwannomas (VS). Although there is nearly 100% penetrance by 60 years of age, some patients suffer from a severe form of the disease and develop multiple tumors at an early age, while others are asymptomatic until later in life. Management options for VS include surgery, stereotactic radiation, and observation with serial imaging; however, currently, there are no FDA-approved pharmacotherapies for NF2 or VS. Recent advancements in the molecular biology underlying NF2 have led to a better understanding of the etiology and pathogenesis of VS. These novel signaling pathways may be used to identify targeted therapies for these tumors. This review discusses the clinical features and treatment options for sporadic- and NF2-associated VS, the diagnostic and screening criteria, completed and ongoing clinical trials, quality of life metrics, and opportunities for future research.

  View details for DOI 10.1093/noajnl/vdaa153

  View details for PubMedID 33604573

  View details for PubMedCentralID PMC7881257

• Altered expression of genes regulating inflammation and synaptogenesis during regrowth of afferent neurons to cochlear hair cells PLOS ONE Wu, C., Brugeaud, A., Seist, R., Lin, H., Yeh, W., Petrillo, M., Coppola, G., Edge, A. B., Stankovic, K. M. 2020; 15 (10): e0238578

  Abstract

  The spiral ganglion neurons constitute the primary connection between auditory hair cells and the brain. The spiral ganglion afferent fibers and their synapse with hair cells do not regenerate to any significant degree in adult mammalian ears after damage. We have investigated gene expression changes after kainate-induced disruption of the synapses in a neonatal cochlear explant model in which peripheral fibers and the afferent synapse do regenerate. We compared gene expression early after damage, during regeneration of the fibers and synapses, and after completion of in vitro regeneration. These analyses revealed a total of 2.5% differentially regulated transcripts (588 out of 24,000) based on a threshold of p<0.005. Inflammatory response genes as well as genes involved in regeneration of neural circuits were upregulated in the spiral ganglion neurons and organ of Corti, where the hair cells reside. Prominent genes upregulated at several time points included genes with roles in neurogenesis (Elavl4 and Sox21), neural outgrowth (Ntrk3 and Ppp1r1c), axonal guidance (Rgmb and Sema7a), synaptogenesis (Nlgn2 and Psd2), and synaptic vesicular function (Syt8 and Syn1). Immunohistochemical and in situ hybridization analysis of genes that had not previously been described in the cochlea confirmed their cochlear expression. The time course of expression of these genes suggests that kainate treatment resulted in a two-phase response in spiral ganglion neurons: an acute response consistent with inflammation, followed by an upregulation of neural regeneration genes. Identification of the genes activated during regeneration of these fibers suggests candidates that could be targeted to enhance regeneration in adult ears.

  View details for DOI 10.1371/journal.pone.0238578

  View details for Web of Science ID 000590270000017

  View details for PubMedID 33001981

  View details for PubMedCentralID PMC7529247

• Cigarette Smoking, Smoking Cessation, and Risk of Hearing Loss in Women AMERICAN JOURNAL OF MEDICINE Lin, B. M., Wang, M., Stankovic, K. M., Eavey, R., McKenna, M. J., Curhan, G. C., Curhan, S. G. 2020; 133 (10): 1180-1186

  Abstract

  Previous studies demonstrated higher risk of hearing loss among cigarette smokers, but longitudinal data on whether the risk is influenced by smoking cessation are limited. We prospectively investigated relations between smoking, smoking cessation, and risk of self-reported moderate or worse hearing loss among 81,505 women in the Nurses' Health Study II (1991-2013).Information on smoking and hearing status was obtained from validated biennial questionnaires. Cox proportional hazards regression was used to estimate multivariable-adjusted relative risks (MVRR, 95% confidence interval).During 1,533,214 person-years of follow-up, 2760 cases of hearing loss were reported. Smoking was associated with higher risk of hearing loss and the risk tended to be higher with greater number of pack-years smoked. Compared with never smokers, the MVRR (95% confidence interval) among past smokers with 20+ pack-years of smoking was 1.30 (1.09-1.55) and 1.21 (1.02-1.43) for current smokers. The magnitude of elevated risk diminished with greater time since smoking cessation. Compared with never smokers, the MVRR among smokers who quit <5 years prior was 1.43 (1.17-1.75); 5-9 years prior was 1.27 (1.03-1.56); 10-14 years prior was 1.17 (0.96-1.41); and plateaued thereafter. Additional adjustment for pack-years smoking attenuated the results.The higher risk of hearing loss associated with smoking may diminish over time after quitting.

  View details for DOI 10.1016/j.amjmed.2020.03.049

  View details for Web of Science ID 000577506000043

  View details for PubMedID 32387319

  View details for PubMedCentralID PMC7541613

• Progression of Contralateral Hearing Loss in Patients With Sporadic Vestibular Schwannoma FRONTIERS IN NEUROLOGY Early, S., Rinnooy Kan, C. E., Eggink, M., Frijns, J. M., Stankovic, K. M. 2020; 11: 796

  Abstract

  Background and Introduction: Vestibular schwannomas (VSs) are the most common tumors of the cerebellopontine angle, typically presenting unilaterally with ipsilateral sensorineural hearing loss (SNHL). The mechanism of tumor-induced hearing loss has recently been shown to be related to secreted tumor factors, in addition to mechanical compression of the adjacent auditory nerve, and these factors may percolate through CSF or blood to affect contralateral hearing as well. Methods: This is a retrospective study of medical records for patients treated for VS at Mass Eye and Ear from January 1994 through October 2018. Included patients had unilateral VS and sequential audiometry allowing for longitudinal assessment of hearing over time. Mass Eye and Ear's audiology database was used to select age- and sex-matched case controls, also with sequential audiometry, from the non-VS population. Subgroup analysis was performed by age, sex, baseline hearing, and tumor size at initial diagnosis. Hearing loss progression was performed using Kaplan-Meier analysis to account for variable follow-up times. Results: A total of 661 patients were identified with VS and sequential audiometry. The population was predominantly female vs. male (368 vs. 293, p = 0.0035), driven primarily by younger patients with Koos 4 tumors (76 female vs. 49 male, p = 0.016). Patients with normal baseline hearing bilaterally (N = 241) demonstrated no significant difference in hearing loss progression in VS-contralateral vs. control ears. Patients with abnormal baseline VS-ipsilateral hearing (N = 190), however, demonstrated significantly higher likelihood of reaching moderate SNHL in VS-contralateral ears. Subgroup analysis by age, sex, and baseline tumor size did not yield any subgroup-specific trends for hearing loss progression. Discussion and Conclusion: This is the largest study to date tracking long-term bilateral hearing outcomes in patients with VS, and demonstrates that, in patients with abnormal hearing in the VS-ipsilateral ear, there exists a long-term risk of progression to moderate hearing loss in the contralateral ear as well. Combined with the absence of significant changes in word understanding in the affected ears, these findings may provide clues to the nature of tumor-secreted factors involved in VS-associated hearing loss. Female predominance within the VS patient population is confirmed, driven mostly by younger female patients with Koos 4 tumors.

  View details for DOI 10.3389/fneur.2020.00796

  View details for Web of Science ID 000566244800001

  View details for PubMedID 33013614

  View details for PubMedCentralID PMC7461819

• MMP-14 (MT1-MMP) Is a Biomarker of Surgical Outcome and a Potential Mediator of Hearing Loss in Patients With Vestibular Schwannomas. Frontiers in cellular neuroscience Ren, Y., Hyakusoku, H., Sagers, J. E., Landegger, L. D., Welling, D. B., Stankovic, K. M. 2020; 14: 191

  Abstract

  Improved biomarkers are needed for vestibular schwannoma (VS), the most common tumor of the cerebellopontine angle, as existing clinical biomarkers have poor predictive value. Factors such as tumor size or growth rate do not shed light on the pathophysiology of associated sensorineural hearing loss (SNHL) and suffer from low specificity and sensitivity, whereas histological markers only sample a fraction of the tumor and are difficult to ascertain before tumor treatment or surgical intervention. Proteases play diverse and critical roles in tumorigenesis and could be leveraged as a new class of VS biomarkers. Using a combination of in silico, in vitro, and ex vivo approaches, we identified matrixmetalloprotease 14 (MMP-14; also known as MT1-MMP), from a panel of candidate proteases that were differentially expressed through the largest meta-analysis of human VS transcriptomes. The abundance and proteolytic activity of MMP-14 in the plasma and tumor secretions from VS patients correlated with clinical parameters and the degree of SNHL. Further, MMP-14 plasma levels correlated with surgical outcomes such as the extent of resection. Finally, the application of MMP-14 at physiologic concentrations to cochlear explant cultures led to damage to spiral ganglion neuronal fibers and synapses, thereby providing mechanistic insight into VS-associated SNHL. Taken together, MMP-14 represents a novel molecular biomarker that merits further validation in both diagnostic and prognostic applications for VS.

  View details for DOI 10.3389/fncel.2020.00191

  View details for PubMedID 32848608

  View details for PubMedCentralID PMC7424165

• Regeneration of Cochlear Synapses by Systemic Administration of a Bisphosphonate FRONTIERS IN MOLECULAR NEUROSCIENCE Seist, R., Tong, M., Landegger, L. D., Vasilijic, S., Hyakusoku, H., Katsumi, S., McKenna, C. E., Edge, A. B., Stankovic, K. M. 2020; 13: 87

  Abstract

  Sensorineural hearing loss (SNHL) caused by noise exposure and attendant loss of glutamatergic synapses between cochlear spiral ganglion neurons (SGNs) and hair cells is the most common sensory deficit worldwide. We show here that systemic administration of a bisphosphonate to mice 24 h after synaptopathic noise exposure regenerated synapses between inner hair cells and SGNs and restored cochlear function. We further demonstrate that this effect is mediated by inhibition of the mevalonate pathway. These results are highly significant because they suggest that bisphosphonates could reverse cochlear synaptopathy for the treatment of SNHL.

  View details for DOI 10.3389/fnmol.2020.00087

  View details for Web of Science ID 000556565200001

  View details for PubMedID 32765216

  View details for PubMedCentralID PMC7381223

• National Trends in Surgical Resection of Vestibular Schwannomas OTOLARYNGOLOGY-HEAD AND NECK SURGERY Ren, Y., Sethi, R. K., Stankovic, K. M. 2020; 163 (6): 1244-1249

  Abstract

  To characterize the national trend for surgical resection of vestibular schwannoma (VS) and to assess changes in demographics, length of stay (LOS), discharge patterns, and hospital charges.Population-based inpatient registry analysis.National Inpatient Sample and SEER database (Surveillance, Epidemiology, and End Results).Retrospective review of the US National Inpatient Sample and the SEER database from 2001 to 2014 of all patients who underwent resection of VS.A total of 24,380 VS resections were performed. While the annual incidence of VS remained stable at 1.38 per 100,000, surgical volume declined by 36.1%, from 2807 in 2001 to 1795 in 2014 (R2 = 0.58). Total hospital charges more than doubled, from $52,475 in 2001 to $115,164 in 2014 ($4478 per year, R2 = 0.96). While most procedures were performed at large-sized hospitals, this decreased from 89% in 2002 to 75.8% in 2014. Average LOS remained stable at 5.2 days during the study period. The number of discharges to a nursing facility increased from 113 (5.5%) in 2002 to 245 (13.6%) in 2014 (P = .0002).VS resection has evolved in the United States. While the incidence remained stable, surgical volume decreased by 36%, and hospital charges more than doubled. More cases are being performed at smaller hospitals. Although LOS did not vary significantly, there is an increase in nonroutine discharges. These data may guide future research in resource utilization in neurotology.

  View details for DOI 10.1177/0194599820932148

  View details for Web of Science ID 000542713400001

  View details for PubMedID 32571146

• Topical fibroblast growth factor-2 for treatment of chronic tympanic membrane perforations LARYNGOSCOPE INVESTIGATIVE OTOLARYNGOLOGY Santos, F., Shu, E., Lee, D. J., Jung, D. H., Quesnel, A., Stankovic, K., Abdul-Aziz, D., Bay, C. P., Quinkert, A., Welling, D. 2020; 5 (4): 657-664

  Abstract

  To determine the efficacy of fibroblast growth factor-2 (FGF-2) in treating chronic nonhealing tympanic membrane (TM) perforations.Double-blinded, randomized placebo controlled phase 2 clinical trial for patients with chronic TM perforations of more than 3 months duration with a cross-over arm. Patients received either FGF-2 or placebo (sterile water) saturated gelatin sponge in the perforation after rimming the perforation under topical anesthesia. The perforation was then covered with Tisseel fibrin glue. The primary endpoint was complete closure of the TM perforation. Secondary end points included change in hearing and partial TM closure rates. The TM was examined every 3 weeks with otoendoscopy for closure. The treatment was repeated if there was incomplete closure every 3 weeks up to a total of three treatments per arm.Seventy four patients were recruited for the study. Fifty seven met eligibility criteria and fifty four completed the study. Ten of 14 perforations closed completely in the placebo group (71.4%) and 23 of 40 perforations closed completely in the FGF-2 treatment group (57.5%), P value = .36. Pure tone averages and word recognition scores were not statistically significantly different between study groups post-treatment. After initial complete closure, re-perforation occurred in seven FGF-2 treated patients and two placebo patients making the effective final closure rate 40% for FGF and 57% for placebo, respectively.No statistically significant difference in tympanic membrane perforation closure rate was found between the FGF-2 and placebo groups. There were no differences in hearing outcomes between the groups.1b.

  View details for DOI 10.1002/lio2.395

  View details for Web of Science ID 000532526900001

  View details for PubMedID 32864435

  View details for PubMedCentralID PMC7444771

• Combination therapy with mTOR kinase inhibitor and dasatinib as a novel therapeutic strategy for vestibular schwannoma SCIENTIFIC REPORTS Sagers, J. E., Beauchamp, R. L., Zhang, Y., Vasilijic, S., Wu, L., DeSouza, P., Seist, R., Zhou, W., Xu, L., Ramesh, V., Stankovic, K. M. 2020; 10 (1): 4211

  Abstract

  Neurofibromatosis type 2 (NF2) is an inherited disorder characterized by bilateral vestibular schwannomas (VS) that arise from neoplastic Schwann cells (SCs). NF2-associated VSs are often accompanied by meningioma (MN), and the majority of NF2 patients show loss of the NF2 tumor suppressor. mTORC1 and mTORC2-specific serum/glucocorticoid-regulated kinase 1 (SGK1) are constitutively activated in MN with loss of NF2. In a recent high-throughput kinome screen in NF2-null human arachnoidal and meningioma cells, we showed activation of EPH RTKs, c-KIT, and SFK members independent of mTORC1/2 activation. Subsequently, we demonstrated in vitro and in vivo efficacy of combination therapy with the dual mTORC1/2 inhibitor AZD2014 and the multi-kinase inhibitor dasatinib. For these reasons, we investigated activated mTORC1/2 and EPH receptor-mediated signaling in sporadic and NF2-associated VS. Using primary human VS cells and a mouse allograft model of schwannoma, we evaluated the dual mTORC1/2 inhibitor AZD2014 and the tyrosine kinase inhibitor dasatinib as monotherapies and in combination. Escalating dose-response experiments on primary VS cells grown from 15 human tumors show that combination therapy with AZD2014 and dasatinib is more effective at reducing metabolic activity than either drug alone and exhibits a therapeutic effect at a physiologically reasonable concentration (~0.1 µM). In vivo, while AZD2014 and dasatinib each inhibit tumor growth alone, the effect of combination therapy exceeds that of either drug. Co-targeting the mTOR and EPH receptor pathways with these or similar compounds may constitute a novel therapeutic strategy for VS, a condition for which there is no FDA-approved pharmacotherapy.

  View details for DOI 10.1038/s41598-020-60156-6

  View details for Web of Science ID 000560099700001

  View details for PubMedID 32144278

  View details for PubMedCentralID PMC7060236

• Intracochlear Perfusion of Tumor Necrosis Factor-Alpha Induces Sensorineural Hearing Loss and Synaptic Degeneration in Guinea Pigs FRONTIERS IN NEUROLOGY Katsumi, S., Sahin, M., Lewis, R. M., Iyer, J. S., Landegger, L. D., Stankovic, K. M. 2020; 10: 1353

  Abstract

  Tumor necrosis factor-alpha (TNF-α) is a proinflammatory cytokine that plays a prominent role in the nervous system, mediating a range of physiologic and pathologic functions. In the auditory system, elevated levels of TNF-α have been implicated in several types of sensorineural hearing loss, including sensorineural hearing loss induced by vestibular schwannoma, a potentially fatal intracranial tumor that originates from the eighth cranial nerve; however, the mechanisms underlying the tumor's deleterious effects on hearing are not well-understood. Here, we investigated the effect of acute elevations of TNF-α in the inner ear on cochlear function and morphology by perfusing the cochlea with TNF-α in vivo in guinea pigs. TNF-α perfusion did not significantly change thresholds for compound action potential (CAP) responses, which reflect cochlear nerve activity, or distortion product otoacoustic emissions, which reflect outer hair cell integrity. However, intracochlear TNF-α perfusion reduced CAP amplitudes and increased the number of inner hair cell synapses without paired post-synaptic terminals, suggesting a pattern of synaptic degeneration that resembles that observed in primary cochlear neuropathy. Additionally, etanercept, a TNF-α blocker, protected against TNF-α-induced synaptopathy when administered systemically prior to intracochlear TNF-α perfusion. Findings motivate further investigation into the harmful effects of chronically elevated intracochlear levels of TNF-α, and the potential for etanercept to counter these effects.

  View details for DOI 10.3389/fneur.2019.01353

  View details for Web of Science ID 000517310700001

  View details for PubMedID 32116980

  View details for PubMedCentralID PMC7025643

• Light-Based Neuronal Activation The Future of Cranial Nerve Stimulation OTOLARYNGOLOGIC CLINICS OF NORTH AMERICA Kozin, E. D., Brown, M., Lee, D. J., Stankovic, K. M. 2020; 53 (1): 171-+

  Abstract

  Despite advances in implant hardware, neuroprosthetic devices in otolaryngology have sustained evolutionary rather than revolutionary changes over the past half century. Although electrical stimulation has the capacity for facile activation of neurons and high temporal resolution, it has limited spatial selectivity. Alternative strategies for neuronal stimulation are being investigated to improve spatial resolution. In particular, light-based neuronal stimulation is a viable alternative and complement to electrical stimulation. This article provides a broad overview of light-based neuronal stimulation technologies. Specific examples of active research on light-based prostheses, including cochlear implants, auditory brainstem implants, retinal implants, and facial nerve implants, are reviewed.

  View details for DOI 10.1016/j.otc.2019.09.011

  View details for Web of Science ID 000501405200015

  View details for PubMedID 31739905

• Imaging hair cells through laser-ablated cochlear bone BIOMEDICAL OPTICS EXPRESS Romito, M., Pu, Y., Stankovic, K. M., Psaltis, D. 2019; 10 (11): 5974-5988

  Abstract

  We report an innovative technique for the visualization of cells through an overlying scattering medium by combining femtosecond laser bone ablation and two-photon excitation fluorescence (TPEF) microscopy. We demonstrate the technique by imaging hair cells in an intact mouse cochlea ex vivo. Intracochlear imaging is important for the assessment of hearing disorders. However, the small size of the cochlea and its encasement in the densest bone in the body present challenging obstacles, preventing the visualization of the intracochlear microanatomy using standard clinical imaging modalities. The controlled laser ablation reduces the optical scattering of the cochlear bone while the TPEF allows visualization of individual cells behind the bone. We implemented optical coherence tomography (OCT) simultaneously with the laser ablation to enhance the precision of the ablation and prevent inadvertent damage to the cells behind the bone.

  View details for DOI 10.1364/BOE.10.005974

  View details for Web of Science ID 000493997700035

  View details for PubMedID 31799058

  View details for PubMedCentralID PMC6865115

• Cochlear histopathology in human genetic hearing loss: State of the science and future prospects HEARING RESEARCH Bommakanti, K., Lyer, J. S., Stankovic, K. M. 2019; 382: 107785

  Abstract

  Sensorineural hearing loss (SNHL) is an extraordinarily common disability, affecting 466 million people across the globe. Half of these incidents are attributed to genetic mutations that disrupt the structure and function of the cochlea. The human cochlea's interior cannot be imaged or biopsied without damaging hearing; thus, everything known about the morphologic correlates of hereditary human deafness comes from histopathologic studies conducted in either cadaveric human temporal bone specimens or animal models of genetic deafness. The purpose of the present review is to a) summarize the findings from all published histopathologic studies conducted in human temporal bones with known SNHL-causing genetic mutations, and b) compare the reported phenotypes of human vs. mouse SNHL caused by the same genetic mutation. The fact that human temporal bone histopathologic analysis has been reported for only 22 of the nearly 200 identified deafness-causing genes suggests a great need for alternative and improved techniques for studying human hereditary deafness; in light of this, the present review concludes with a summary of promising future directions, specifically in the fields of high resolution cochlear imaging, intracochlear fluid biopsy, and gene therapy.

  View details for DOI 10.1016/j.heares.2019.107785

  View details for Web of Science ID 000491214200002

  View details for PubMedID 31493568

  View details for PubMedCentralID PMC6778517

• NLRP3 inflammasome activation in human vestibular schwannoma: Implications for tumor-induced hearing loss HEARING RESEARCH Sagers, J. E., Sahin, M. I., Moon, I., Ahmed, S. G., Stemmer-Rachamimov, A., Brenner, G. J., Stankovic, K. M. 2019; 381: 107770

  Abstract

  Vestibular schwannoma (VS) is the fourth most common intracranial tumor, arising from neoplastic Schwann cells of the vestibular nerve and often causing debilitating sensorineural hearing loss (SNHL) and tinnitus. Previous research suggests that the abnormal upregulation of inflammatory pathways plays a highly significant, though infrequently described role in VS pathobiology, and that VS-associated SNHL is due not only to mechanical compression of the auditory nerve but also to differences in the intrinsic biology of these tumors. We hypothesize that patients who present with poor hearing associated with VS experience a more robust inflammatory response to this tumor than VS patients who present with good hearing. To investigate this hypothesis, we conducted a comprehensive pathway analysis using gene expression data from the largest meta-analysis of vestibular schwannoma microarray data, comprising 80 tumors and 16 healthy peripheral nerves. We identified the NLRP3 inflammasome as a novel target worthy of further exploration in VS research and validated this finding at the gene and protein expression level in human VS tissue using qRT-PCR and immunohistochemistry. To date, NLRP3 inflammasome activation has not been reported in VS, and this finding may represent a new and potentially significant therapeutic avenue. Notably, after analysis of 30 VSs, we observe that overexpression of key components of the NLRP3 inflammasome is preferentially associated with tumors that produce increased hearing loss in VS patients. Therefore, therapeutic development for VS should include considerations for minimizing NLRP3-associated inflammation to best preserve hearing.

  View details for DOI 10.1016/j.heares.2019.07.007

  View details for Web of Science ID 000487170200001

  View details for PubMedID 31430634

• A novel microneedle device for controlled and reliable liquid biopsy of the human inner ear HEARING RESEARCH Early, S., Moon, I., Bommakanti, K., Hunter, I., Stankovic, K. M. 2019; 381: 107761

  Abstract

  Sensorineural hearing loss is the most common sensory deficit worldwide, yet our understanding of the underlying pathophysiology is limited by the challenges of access to the inner ear in a safe and reliable manner. We present a novel microneedle device for trans-round window membrane liquid biopsy, which utilizes controlled depth of perforation and microliter aspiration control to safely biopsy fluids of the inner ear. Of eleven devices tested in fresh frozen human temporal bones, seven demonstrated alignment between electrical, visual and tactile detection of round window membrane perforation, and nine were successful in aspiration of meaningful diagnostic samples from the perilymphatic space. Purity of the average perilymph sample was 69% for a 5 μL sample volume, equivalent to 3.5 μL attributable to perilymph. Diagnostic success was shown both by transmastoid facial recess and transcanal tympanotomy approach. This device can enable new advances in the understanding of inner ear pathology, and brings us one step closer to liquid biopsy of the inner ear becoming a routine part of clinical care.

  View details for DOI 10.1016/j.heares.2019.06.004

  View details for Web of Science ID 000487170200016

  View details for PubMedID 31353070

• Cytokine Levels in Inner Ear Fluid of Young and Aged Mice as Molecular Biomarkers of Noise-Induced Hearing Loss FRONTIERS IN NEUROLOGY Landegger, L. D., Vasilijic, S., Fujita, T., Soares, V. Y., Seist, R., Xu, L., Stankovic, K. M. 2019; 10: 977

  Abstract

  Sensorineural hearing loss (SNHL) is the most common sensory deficit worldwide, frequently caused by noise trauma and aging, with inflammation being implicated in both pathologies. Here, we provide the first direct measurements of proinflammatory cytokines in inner ear fluid, perilymph, of adolescent and 2-year-old mice. The perilymph of adolescent mice exposed to the noise intensity resulting in permanent auditory threshold elevations had significantly increased levels of IL-6, TNF-α, and CXCL1 6 h after exposure, with CXCL1 levels being most elevated (19.3 ± 6.2 fold). We next provide the first immunohistochemical localization of CXCL1 in specific cochlear supporting cells, and its presumed receptor, Duffy antigen receptor for chemokines (DARC), in hair cells and spiral ganglion neurons. Our results demonstrate the feasibility of molecular diagnostics of SNHL using only 0.5 μL of perilymph, and motivate future sub-μL based diagnostics of human SNHL based on liquid biopsy of the inner ear to guide therapy, promote hearing protection, and monitor response to treatment.

  View details for DOI 10.3389/fneur.2019.00977

  View details for Web of Science ID 000485185600001

  View details for PubMedID 31632328

  View details for PubMedCentralID PMC6749100

• Gene Therapy for Human Sensorineural Hearing Loss FRONTIERS IN CELLULAR NEUROSCIENCE Ren, Y., Landegger, L. D., Stankovic, K. M. 2019; 13: 323

  Abstract

  Hearing loss is the most common sensory impairment in humans and currently disables 466 million people across the world. Congenital deafness affects at least 1 in 500 newborns, and over 50% are hereditary in nature. To date, existing pharmacologic therapies for genetic and acquired etiologies of deafness are severely limited. With the advent of modern sequencing technologies, there is a vast compendium of growing genetic alterations that underlie human hearing loss, which can be targeted by therapeutics such as gene therapy. Recently, there has been tremendous progress in the development of gene therapy vectors to treat sensorineural hearing loss (SNHL) in animal models in vivo. Nevertheless, significant hurdles remain before such technologies can be translated toward clinical use. These include addressing the blood-labyrinth barrier, engineering more specific and effective delivery vehicles, improving surgical access, and validating novel targets. In this review, we both highlight recent progress and outline challenges associated with in vivo gene therapy for human SNHL.

  View details for DOI 10.3389/fncel.2019.00323

  View details for Web of Science ID 000475960300001

  View details for PubMedID 31379508

  View details for PubMedCentralID PMC6660246

• Gene therapy with apoptosis-associated speck-like protein, a newly described schwannoma tumor suppressor, inhibits schwannoma growth in vivo. Neuro-oncology Ahmed, S. G., Abdelnabi, A., Maguire, C. A., Doha, M., Sagers, J. E., Lewis, R. M., Muzikansky, A., Giovannini, M., Stemmer-Rachamimov, A., Stankovic, K. M., Fulci, G., Brenner, G. J. 2019; 21 (7): 854-866

  Abstract

  We evaluated apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) as a schwannoma tumor suppressor and explored its utilization in a schwannoma gene therapy strategy that may be translated to clinical use.ASC protein expression and mRNA level were assessed in human schwannoma by immunohistochemistry and quantitative PCR, respectively. Methylation- specific PCR was used to assess ASC promoter methylation. The effect of ASC overexpression in schwannoma cells was evaluated through ATP-based viability, lactate dehydrogenase release, and apoptosis staining. Western blotting and colorimetric assay were used to test the effect of ASC overexpression on endogenous pro-apoptotic pathways. Bioluminescence imaging, behavioral testing, and immunohistochemistry in human xenograft and murine allograft schwannoma models were used to examine the efficacy and toxicity of intratumoral injection of adeno-associated virus (AAV) vector encoding ASC.ASC expression was suppressed via promoter methylation in over 80% of the human schwannomas tested. ASC overexpression in schwannoma cells results in cell death and is associated with activation of endogenous caspase-9, caspase-3, and upregulation of BH3 interacting-domain death agonist. In a human xenograft schwannoma model, AAV1-mediated ASC delivery reduced tumor growth and resolved tumor-associated pain without detectable toxicity, and tumor control was associated with reduced Ki67 mitotic index and increased tumor-cell apoptosis. Efficacy of this schwannoma gene therapy strategy was confirmed in a murine schwannoma model.We have identified ASC as a putative schwannoma tumor suppressor with high potential clinical utility for schwannoma gene therapy and generated a vector that treats schwannomas via a novel mechanism that does not overlap with current treatments.

  View details for DOI 10.1093/neuonc/noz065

  View details for PubMedID 30977509

  View details for PubMedCentralID PMC6620641

• Proteome of normal human perilymph and perilymph from people with disabling vertigo. PloS one Lin, H. C., Ren, Y., Lysaght, A. C., Kao, S. Y., Stankovic, K. M. 2019; 14 (6): e0218292

  Abstract

  The vast majority of hearing loss, the most common sensory impairment, and vertigo, which commonly causes falls, both reflect underlying dysfunction of inner ear cells. Perilymph sampling can thus provide molecular cues to hearing and balance disorders. While such "liquid biopsy" of the inner ear is not yet in routine clinical practice, previous studies have uncovered alterations in perilymph in patients with certain types of hearing loss. However, the proteome of perilymph from patients with intact hearing has been unknown. Furthermore, no complete characterization of perilymph from patients with vestibular dysfunction has been reported. Here, using liquid-chromatography with tandem mass spectrometry, we analyzed samples of normal perilymph collected from three patients with skull base meningiomas and intact hearing. We identified 228 proteins that were common across the samples, establishing a greatly expanded proteome of the previously inferred normal human perilymph. Further comparison to perilymph obtained from three patients with vestibular dysfunction with drop attacks due to Meniere's disease showed 38 proteins with significantly differential abundance. The abundance of four protein candidates with previously unknown roles in inner ear biology was validated in murine cochleae by immunohistochemistry and in situ hybridization: AACT, HGFAC, EFEMP1, and TGFBI. Together, these results motivate future work in characterizing the normal human perilymph and identifying biomarkers of inner ear disease.

  View details for DOI 10.1371/journal.pone.0218292

  View details for PubMedID 31185063

  View details for PubMedCentralID PMC6559673

• Periostin and Inflammatory Disease: Implications for Chronic Rhinosinusitis OTOLARYNGOLOGY-HEAD AND NECK SURGERY Lehmann, A. E., Scangas, G. A., Bergmark, R. W., El Rassi, E., Stankovic, K. M., Metson, R. 2019; 160 (6): 965-973

  Abstract

  To provide a comprehensive overview of the emerging role of periostin, an extracellular matrix protein, as a key component in the development, diagnosis, and treatment of patients with chronic rhinosinusitis.Medline database.A state of the art review was performed targeting English-language studies investigating the role of periostin in cardiopulmonary, neoplastic, and inflammatory diseases, with emphasis on recent advances in the study of periostin in chronic rhinosinusitis.Periostin has emerged as a novel biomarker and therapeutic target for numerous human pathologies, including cardiac, pulmonary, and neoplastic disease. The upregulation of periostin in chronic rhinosinusitis suggests the potential for similar roles among patients with sinonasal disease.Chronic rhinosinusitis is a widespread disease with major clinical and societal impact. A critical limitation in the current treatment of patients with chronic rhinosinusitis is the absence of clinically relevant biomarkers to guide diagnosis and treatment selection. A review of the literature supports a likely role of periostin as a biomarker of chronic rhinosinusitis, as well as a novel therapeutic target in the future treatment of patients with sinonasal disease.

  View details for DOI 10.1177/0194599819838782

  View details for Web of Science ID 000470767300004

  View details for PubMedID 30935271

• A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma. Nature protocols Chen, J., Landegger, L. D., Sun, Y., Ren, J., Maimon, N., Wu, L., Ng, M. R., Chen, J. W., Zhang, N., Zhao, Y., Gao, X., Fujita, T., Roberge, S., Huang, P., Jain, R. K., Plotkin, S. R., Stankovic, K. M., Xu, L. 2019; 14 (2): 541-555

  Abstract

  Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region. We also describe how to apply state-of-the-art intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor-stroma interaction assays can be applied, and the model can be used to test the efficacy of novel therapeutic approaches. By studying the disease from every angle, this model offers the potential to unravel the basic biological underpinnings of NF2 and to develop novel therapeutics to control this devastating disease. Our protocol can be adapted to study other diseases within the CPA, including meningiomas, lipomas, vascular malformations, hemangiomas, epidermoid cysts, cerebellar astrocytomas, and metastatic lesions. The entire surgical procedure takes ~45 min per mouse and allows for subsequent longitudinal imaging, as well as neurological and hearing assessment, for up to 2 months.

  View details for DOI 10.1038/s41596-018-0105-7

  View details for PubMedID 30617350

  View details for PubMedCentralID PMC6571021

• Fracture of the Incus Caused by Digital Manipulation of the Ear Canal and its Diagnosis Using Wideband Acoustic Immittance OTOLOGY & NEUROTOLOGY Masud, S. F., Knudson, I. M., Stankovic, K. M., Nakajima, H. 2019; 40 (2): E115-E118

  Abstract

  To describe the first reported case of a fracture of the long process of the incus due to digital manipulation of the ear canal and to discuss diagnostic markers for ossicular fractures.A 46-year-old woman with incessant clicking and crunching in her left ear, and hearing loss after digital manipulation of the ear canal.Diagnostic evaluation and therapeutic ossiculoplasty.Audiometric and wideband acoustic immittance (WAI) measurements were made before surgery to investigate the cause of clicking sounds and mild conductive hearing loss (CHL).The clinical suspicion of a loose ossicular chain was confirmed by a large narrow-band decrease in power reflectance (calculated from WAI) at frequencies between 600 and 700 Hz, and a mid- to high-frequency air-bone gap. Exploratory tympanotomy revealed an ossicular fracture of the distal aspect of the long process of the incus. Ossiculoplasty with bone cement resolved bothersome clicking sounds.A finger inserted into the ear canal can produce an air seal, and subsequent quick removal of the finger can result in the fracture of an ossicle. Clinicians should be cognizant of this form of trauma because insertion of a finger, ear plug, and earphone into the ear canal are common. Ossicular fractures can result in high-frequency CHL, and can be misdiagnosed as sensorineural loss because bone conduction thresholds are not measured above 4 kHz. As in this case, an ossicular fracture may be misdiagnosed and result in inappropriate treatment. Here, WAI, a non-invasive measure of ear mechanics, diagnosed a loose ossicular chain.

  View details for DOI 10.1097/MAO.0000000000002103

  View details for Web of Science ID 000457785100008

  View details for PubMedID 30624402

• Selective femtosecond laser ablation via two-photon fluorescence imaging through a multimode fiber BIOMEDICAL OPTICS EXPRESS Kakkava, E., Romito, M., Conkey, D. B., Loterie, D., Stankovic, K. M., Moser, C., Psaltis, D. 2019; 10 (2): 423-433

  Abstract

  We demonstrate the ability of a multimode fiber probe to provide two-photon fluorescence (TPF) imaging feedback that guides the femtosecond laser ablation (FLA) in biological samples for highly selective modifications. We implement the system through the propagation of high power femtosecond pulses through a graded-index (GRIN) multimode fiber and we investigate the limitations posed by the high laser peak intensities required for laser ablation. We demonstrate that the GRIN fiber probe can deliver laser intensities up to 1.5x1013 W/cm2, sufficient for the ablation of a wide range of materials, including biological samples. Wavefront shaping through an ultrathin probe of around 400 μm in diameter is used for diffraction limited focusing and digital scanning of the focus spot. Selective FLA of cochlear hair cells is performed based on the TPF images obtained through the same multimode fiber probe.

  View details for DOI 10.1364/BOE.10.000423

  View details for Web of Science ID 000457500300004

  View details for PubMedID 30800490

  View details for PubMedCentralID PMC6377891

• Acute Otitis Media and Associated Complications in United States Emergency Departments Ren, Y., Sethi, R. V., Stankovic, K. M. LIPPINCOTT WILLIAMS & WILKINS. 2018: 1005-1011

  Abstract

  Complications due to acute otitis media (AOM), while rare, are associated with significant morbidity and are not well characterized from an epidemiological perspective. We analyze the pattern of presentation and emergency department (ED) utilization in patients with AOM and associated complications.Retrospective analysis of the Nationwide Emergency Department Sample (NEDS) from 2009 to 2011.Emergency Department.Patients who presented with a primary diagnosis of AOM or acute mastoiditis.Diagnostic.NEDS was queried for patient encounters with a diagnosis of AOM or acute mastoiditis based on ICD-9 codes. Complications of severe infection, including petrositis, Gradenigo's syndrome, facial paresis, labyrinthitis, meningitis, intracranial abscess, venous sinus thrombosis, and cerebrospinal fluid leak, were assessed. Weighted estimates for demographics, types of complications, socioeconomic status, and trends over time were extracted.A weighted total of 5,811,127 ED visits were identified. The majority of patients were less than 18 years old (79.9%) with an average age of 10.1 years. Most were discharged (99.4%). There were 15,243 (0.26%) patients who presented with a complication. The most common complications were acute mastoiditis (0.16%), labyrinthitis (0.06%), and facial paresis (0.03%). Compared with patients with uncomplicated AOM, patients with complicated AOM were older (37 vs. 10-yr old), insured by Medicare (18% vs. 2.1%), and more likely to be admitted (43.6% vs. 0.4%) (p < 0.0001, for all comparisons).ED visits related to AOM or mastoiditis are common and complications are rare. An in-depth analysis on a national level is useful for assessing healthcare utilization trends.

  View details for DOI 10.1097/MAO.0000000000001929

  View details for Web of Science ID 000457777400033

  View details for PubMedID 30113560

  View details for PubMedCentralID PMC6097248

• Noncontrast vestibular schwannoma surveillance imaging including an MR cisternographic sequence: is there a need for postcontrast imaging? Journal of neurosurgery Buch, K., Juliano, A., Stankovic, K. M., Curtin, H. D., Cunnane, M. B. 2018; 131 (2): 549-554

  Abstract

  The purpose of this study was to evaluate the use of a noncontrast MRI protocol that includes a cisternographic sequence (CISS/FIESTA/3D DRIVE) compared to a protocol that includes a gadolinium-enhanced sequence in order to determine whether a noncontrast approach could be utilized to follow vestibular schwannomas.A total of 251 patients with vestibular schwannomas who underwent MRI of the temporal bones that included both cisternographic sequence and postcontrast T1 imaging between January 2000 and January 2016 for surveillance were included in this retrospective study. The size of the vestibular schwannomas was independently assessed on a noncontrast MR cisternographic sequence and compared to size measurements on a postcontrast sequence. The evaluation of intralesional cystic components (identified as T2 signal hyperintensity) and hemorrhagic components (identified with intrinsic T1 hyperintensity) on noncontrast MR sequences was compared to evaluation on postcontrast MR sequences to determine whether additional information could be derived from the postcontrast sequences. Additionally, any potentially clinically significant, incidentally detected findings on the postcontrast T1 sequences were documented and compared with the detection of these findings on the precontrast images.No significant difference in vestibular schwannoma size was found when comparing measurements made on the images obtained with the MR cisternographic sequence and those made on images obtained with the postcontrast sequence (p = 0.99). Noncontrast MR images were better (detection rate of 87%) than postcontrast images for detection of cystic components. Noncontrast MR images were also better for identifying hemorrhagic components. No additional clinically relevant information regarding the tumors was identified on the postcontrast sequences.Based on the results of this study, a noncontrast MR protocol that includes a cisternographic sequence would be sufficient for the accurate characterization of size and signal characteristics of vestibular schwannomas, obviating the need for gadolinium contrast administration for the routine surveillance of these lesions.

  View details for DOI 10.3171/2018.3.JNS1866

  View details for PubMedID 30095338

• Ancestral Adeno-Associated Virus Vector Delivery of Opsins to Spiral Ganglion Neurons: Implications for Optogenetic Cochlear Implants. Molecular therapy : the journal of the American Society of Gene Therapy Duarte, M. J., Kanumuri, V. V., Landegger, L. D., Tarabichi, O., Sinha, S., Meng, X., Hight, A. E., Kozin, E. D., Stankovic, K. M., Brown, M. C., Lee, D. J. 2018; 26 (8): 1931-1939

  Abstract

  Optogenetics is a transformative technology based on light-sensitive microbial proteins, known as opsins, that enable precise modulation of neuronal activity with pulsed radiant energy. Optogenetics has been proposed as a means to improve auditory implant outcomes by reducing channel interaction and increasing electrode density, but the introduction of opsins into cochlear spiral ganglion neurons (SGNs) in vivo has been challenging. Here we test opsin delivery using a synthetically developed ancestral adeno-associated virus (AAV) vector called Anc80L65. Wild-type C57BL/6 mouse pups were injected via the round window of cochlea with Anc80L65 carrying opsin Chronos under the control of a CAG promoter. Following an incubation of 6-22 weeks, pulsed blue light was delivered to cochlear SGNs via a cochleosotomy approach and flexible optical fiber. Optically evoked auditory brainstem responses (oABRs) and multiunit activity in inferior colliculus (IC) were observed. Post-experiment cochlear histology demonstrated opsin expression in SGNs (mean = 74%), with an even distribution of opsin along the cochlear basal/apical gradient. This study is the first to describe robust SGN transduction, opsin expression, and optically evoked auditory electrophysiology in neonatal mice. Ultimately, this work may provide the basis for a new generation of cochlear implant based on light.

  View details for DOI 10.1016/j.ymthe.2018.05.023

  View details for PubMedID 30017876

  View details for PubMedCentralID PMC6094394

• Visualizing the 3D cytoarchitecture of the human cochlea in an intact temporal bone using synchrotron radiation phase contrast imaging BIOMEDICAL OPTICS EXPRESS Iyer, J. S., Zhu, N., Gasilov, S., Ladak, H. M., Agrawal, S. K., Stankovic, K. M. 2018; 9 (8): 3757-3767

  Abstract

  The gold standard method for visualizing the pathologies underlying human sensorineural hearing loss has remained post-mortem histology for over 125 years, despite awareness that histological preparation induces severe artifacts in biological tissue. Historically, the transition from post-mortem assessment to non-invasive clinical biomedical imaging in living humans has revolutionized diagnosis and treatment of disease; however, innovation in non-invasive techniques for cellular-level intracochlear imaging in humans has been difficult due to the cochlea's small size, complex 3D configuration, fragility, and deep encasement within bone. Here we investigate the ability of synchrotron radiation-facilitated X-ray absorption and phase contrast imaging to enable visualization of sensory cells and nerve fibers in the cochlea's sensory epithelium in situ in 3D intact, non-decalcified, unstained human temporal bones. Our findings show that this imaging technique resolves the bone-encased sensory epithelium's cytoarchitecture with unprecedented levels of cellular detail for an intact, unstained specimen, and is capable of distinguishing between healthy and damaged epithelium. All analyses were performed using commercially available software that quickly reconstructs and facilitates 3D manipulation of massive data sets. Results suggest that synchrotron radiation phase contrast imaging has the future potential to replace histology as a gold standard for evaluating intracochlear structural integrity in human specimens, and motivate further optimization for translation to the clinic.

  View details for DOI 10.1364/BOE.9.003757

  View details for Web of Science ID 000440490500021

  View details for PubMedID 30338153

  View details for PubMedCentralID PMC6191620

• EMG-based Real Time Facial Gesture Recognition for Stress Monitoring. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Annual International Conference Orguc, S., Khurana, H. S., Stankovic, K. M., Leel, H. S., Chandrakasan, A. P. 2018; 2018: 2651-2654

  Abstract

  An electromyogram (EMG) signal acquisition system capable of real time classification of several facial gestures is presented. The training data consist of the facial EMG collected from 10 individuals (5 female/5 male). A custom-designed sensor interface integrated circuit (IC) consisting of an amplifier and an ADC, implemented in 65nm CMOS technology, has been used for signal acquisition [1]. It consumes 3.8nW power from a 0.3V battery. Feature extraction and classification is performed in software every 300ms to give real-time feedback to the user. Discrete wavelet transforms (DWT) are used for feature extraction in the time-frequency domain. The dimensionality of the feature vector is reduced by selecting specific wavelet decomposition levels without compromising the accuracy, which reduces the computation cost of feature extraction in embedded implementations. A support vector machine (SVM) is used for the classification. Overall, the system is capable of identifying several jaw movements such as clenching, opening the jaw and resting in real-time from a single channel EMG data, which makes the system suitable for providing biofeedback during sleeping and awake states for stress monitoring, bruxism, and several orthodontic applications such as temporomandibular joint disorder (TMJD).

  View details for DOI 10.1109/EMBC.2018.8512781

  View details for PubMedID 30440952

• Computational repositioning and preclinical validation of mifepristone for human vestibular schwannoma SCIENTIFIC REPORTS Sagers, J. E., Brown, A. S., Vasilijic, S., Lewis, R. M., Sahin, M. I., Landegger, L. D., Perlis, R. H., Kohane, I. S., Welling, D., Patel, C. J., Stankovic, K. M. 2018; 8: 5437

  Abstract

  The computational repositioning of existing drugs represents an appealing avenue for identifying effective compounds to treat diseases with no FDA-approved pharmacotherapies. Here we present the largest meta-analysis to date of differential gene expression in human vestibular schwannoma (VS), a debilitating intracranial tumor, and use these data to inform the first application of algorithm-based drug repositioning for this tumor class. We apply an open-source computational drug repositioning platform to gene expression data from 80 patient tumors and identify eight promising FDA-approved drugs with potential for repurposing in VS. Of these eight, mifepristone, a progesterone and glucocorticoid receptor antagonist, consistently and adversely affects the morphology, metabolic activity, and proliferation of primary human VS cells and HEI-193 human schwannoma cells. Mifepristone treatment reduces VS cell viability more significantly than cells derived from patient meningiomas, while healthy human Schwann cells remain unaffected. Our data recommend a Phase II clinical trial of mifepristone in VS.

  View details for DOI 10.1038/s41598-018-23609-7

  View details for Web of Science ID 000428994100030

  View details for PubMedID 29615643

  View details for PubMedCentralID PMC5882888

• The Role of Tumor Necrosis Factor Alpha (TNFα)in Hearing Loss and Vestibular Schwannomas. Current otorhinolaryngology reports Ren, Y., Stankovic, K. M. 2018; 6 (1): 15-23

  Abstract

  The aim of this review is to highlight relevant literature on the role of tumor necrosis factor alpha (TNFα) in sensorineural hearing loss (SNHL) and vestibular schwannomas (VS).A comprehensive review of publically available databases including PubMed was performed. The mechanism by which hearing loss occurs in VS is still unknown and likely multifactorial. Genetic differences between VSs and tumor secreted proteins may be responsible, at least in part, for VS-associated SNHL. TNFα has pleotropic roles in promoting inflammation, maintaining cellular homeostasis, inducing apoptosis, and mediating ototoxicity in patients with sporadic VS. TNFα-targeted therapies have shown efficacy in both animal models of sensorineural hearing loss and clinical trials in patients with immune-mediated hearing loss. Efforts are underway to develop novel nanotechnology-based methods to target TNFα and other pathogenic molecules in VS.Development of molecularly targeted therapies against TNFα represents an important area of research in ameliorating VS-associated hearing loss.

  View details for DOI 10.1007/s40136-018-0186-4

  View details for PubMedID 31485383

  View details for PubMedCentralID PMC6724722

• Targeting the cMET pathway augments radiation response without adverse effect on hearing in NF2 schwannoma models PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Zhao, Y., Liu, P., Zhang, N., Chen, J., Landegger, L. D., Wu, L., Zhao, F., Zhao, Y., Zhang, Y., Zhang, J., Fujita, T., Stemmer-Rachamimov, A., Ferraro, G. B., Liu, H., Muzikansky, A., Plotkin, S. R., Stankovic, K. M., Jain, R. K., Xu, L. 2018; 115 (9): E2077-E2084

  Abstract

  Neurofibromatosis type II (NF2) is a disease that needs new solutions. Vestibular schwannoma (VS) growth causes progressive hearing loss, and the standard treatment, including surgery and radiotherapy, can further damage the nerve. There is an urgent need to identify an adjunct therapy that, by enhancing the efficacy of radiation, can help lower the radiation dose and preserve hearing. The mechanisms underlying deafness in NF2 are still unclear. One of the major limitations in studying tumor-induced hearing loss is the lack of mouse models that allow hearing testing. Here, we developed a cerebellopontine angle (CPA) schwannoma model that faithfully recapitulates the tumor-induced hearing loss. Using this model, we discovered that cMET blockade by crizotinib (CRZ) enhanced schwannoma radiosensitivity by enhancing DNA damage, and CRZ treatment combined with low-dose radiation was as effective as high-dose radiation. CRZ treatment had no adverse effect on hearing; however, it did not affect tumor-induced hearing loss, presumably because cMET blockade did not change tumor hepatocyte growth factor (HGF) levels. This cMET gene knockdown study independently confirmed the role of the cMET pathway in mediating the effect of CRZ. Furthermore, we evaluated the translational potential of cMET blockade in human schwannomas. We found that human NF2-associated and sporadic VSs showed significantly elevated HGF expression and cMET activation compared with normal nerves, which correlated with tumor growth and cyst formation. Using organoid brain slice culture, cMET blockade inhibited the growth of patient-derived schwannomas. Our findings provide the rationale and necessary data for the clinical translation of combined cMET blockade with radiation therapy in patients with NF2.

  View details for DOI 10.1073/pnas.1719966115

  View details for Web of Science ID 000426152500021

  View details for PubMedID 29440379

  View details for PubMedCentralID PMC5834719

• Periostin as a Biomarker for Nasal Polyps in Chronic Rhinosinusitis OTOLARYNGOLOGY-HEAD AND NECK SURGERY Maxfield, A. Z., Landegger, L. D., Brook, C. D., Lehmann, A. E., Campbell, A. P., Bergmark, R. W., Stankovic, K. M., Metson, R. 2018; 158 (1): 181-186

  Abstract

  Objective Periostin is an extracellular matrix protein that is elevated in the sinonasal tissues of patients with chronic rhinosinusitis (CRS). The purpose of this study was to determine whether serum periostin could serve as a molecular biomarker of nasal polyp burden in sinonasal disease. Study Design Prospective cohort study. Setting Academic medical center. Subjects and Methods Serum periostin levels were measured by ELISA on blood samples collected from patients undergoing sinus surgery for CRS (n = 71), further stratified by phenotype as defined by nasal polyps and asthma. Results were compared with assays performed on control subjects (n = 62). Results Mean serum periostin levels were markedly elevated in patients with CRS versus controls (66.1 ng/mL [95% CI, 51.6-80.6] vs 38.7 ng/mL [95% CI, 34.4-42.9], respectively, P = .004). In addition, mean periostin levels were significantly higher in CRS patients with nasal polyps as compared with those without polyps (94.8 ng/mL [95% CI, 67.3-122.4] vs 41.1 ng/mL [95% CI, 35.2-47.0], respectively, P < .001). Periostin levels did not correlate with sex ( P = .473), smoking history ( P = .748), aspirin-exacerbated respiratory disease status ( P = .136), oral steroid use within 1 month of surgery ( P = .281), use of topical steroid nasal spray ( P = .864), or number of prior sinus operations ( P = .973). Conclusion Serum periostin appears to be a novel molecular biomarker for the presence of nasal polyps and may serve as an indicator of CRS endotypes.

  View details for DOI 10.1177/0194599817737967

  View details for Web of Science ID 000419308700029

  View details for PubMedID 29040053

• Cochlear Dysfunction is not Common in Human Meningioma of the Internal Auditory Canal OTOLOGY & NEUROTOLOGY Landegger, L. D., Lee, J. D., Linthicum, F. H., Stankovic, K. M. 2017; 38 (10): E486-E489

  Abstract

  Cochlear dysfunction is not common in human meningioma of the internal auditory canal.Meningiomas arising from the cerebellopontine angle and internal auditory canal typically cause hearing loss. Cochlear dysfunction is known to contribute to sensorineural hearing loss induced by vestibular schwannoma, the most common tumor of the internal auditory canal. Detailed cochlear histopathology in meningioma has not been reported.Retrospective analysis of cochlear histopathology in five unoperated and five operated meningiomas of the internal auditory canal identified after screening human temporal bone collections from three academic medical centers.While some dysfunction of all analyzed cochlear cell types was identified, a predominant or exclusive loss of hair cells was not observed in any meningioma. Only 14.3% of temporal bones showed significantly more hair cell damage on the side of the tumor when compared with the contralateral ear; cochlear neuronal damage was more prevalent in meningiomas. The incidence of hydrops, perilymphatic precipitate, or endolymphatic precipitate was low.Substantial cochlear damage in human meningioma of the internal auditory canal is not common. This may explain the anecdotal hearing improvement observed after surgical resection of meningioma. Our findings underline the importance of developing therapeutic strategies to prevent cochlear neuronal degeneration due to tumors of the internal auditory canal.

  View details for DOI 10.1097/MAO.0000000000001582

  View details for Web of Science ID 000425284200015

  View details for PubMedID 28984808

  View details for PubMedCentralID PMC8106507

• Energy-efficient waveform for electrical stimulation of the cochlear nerve SCIENTIFIC REPORTS Yip, M., Bowers, P., Noel, V., Chandrakasan, A., Stankovic, K. M. 2017; 7: 13582

  Abstract

  The cochlear implant (CI) is the most successful neural prosthesis, restoring the sensation of sound in people with severe-to-profound hearing loss by electrically stimulating the cochlear nerve. Existing CIs have an external, visible unit, and an internal, surgically-placed unit. There are significant challenges associated with the external unit, as it has limited utility and CI users often report a social stigma associated with prosthesis visibility. A fully-implantable CI (FICI) would address these issues. However, the volume constraint imposed on the FICI requires less power consumption compared to today's CI. Because neural stimulation by CI electrodes accounts for up to 90% of power consumption, reduction in stimulation power will result directly in CI power savings. To determine an energy-efficient waveform for cochlear nerve stimulation, we used a genetic algorithm approach, incorporating a computational model of a single mammalian myelinated cochlear nerve fiber coupled to a stimulator-electrode-tissue interface. The algorithm's prediction was tested in vivo in human CI subjects. We find that implementation of a non-rectangular biphasic neural stimulation waveform may result in up to 25% charge savings and energy savings within the comfortable range of hearing for CI users. The alternative waveform may enable future development of a FICI.

  View details for DOI 10.1038/s41598-017-13671-y

  View details for Web of Science ID 000413191500021

  View details for PubMedID 29051546

  View details for PubMedCentralID PMC5648926

• Tumor-Penetrating Delivery of siRNA against TNFα to Human Vestibular Schwannomas. Scientific reports Ren, Y., Sagers, J. E., Landegger, L. D., Bhatia, S. N., Stankovic, K. M. 2017; 7 (1): 12922

  Abstract

  Vestibular schwannoma (VS) is the most common tumor of the cerebellopontine angle, and it typically presents with sensorineural hearing loss. The genomic landscape of schwannoma is complex and many of the molecules implicated in VS pathogenesis represent targets not amenable to antibody-based or small molecule therapeutics. Tumor-targeted delivery of small interfering RNA (siRNA) therapeutics provides a direct and effective means to interrogate targets while minimizing off-target effects. To establish a preclinical model for therapeutic inhibition of putative targets in VS, archived tumor specimens, fresh tumor cells derived from patients with sporadic VS, and an established schwannoma cell line were screened. Nanoparticles directed by the tumor-homing peptide iRGD were selectively taken up by primary VS cultures in vitro via interactions with αvβ3/β5 integrins and neuropilin-1 (NRP-1). Cellular uptake was inhibited by a neutralizing antibody against αv integrin in a dose-dependent manner. When applied to primary VS cultures, iRGD-targeted nanoparticles delivered siRNA directed against TNFα in a receptor-specific fashion to potently silence gene expression and protein secretion. Taken together, our results provide a proof of principle for tumor-targeted, nanoparticle-mediated delivery of siRNA to VS and establish a novel platform for the development and pre-clinical screening of molecular therapeutics against VS.

  View details for DOI 10.1038/s41598-017-13032-9

  View details for PubMedID 29018206

  View details for PubMedCentralID PMC5635039

• Human Cochlear Histopathology Reflects Clinical Signatures of Primary Neural Degeneration SCIENTIFIC REPORTS Sagers, J. E., Landegger, L. D., Worthington, S., Nadol, J. B., Stankovic, K. M. 2017; 7: 4884

  Abstract

  Auditory neuropathy is a significant and understudied cause of human hearing loss, diagnosed in patients who demonstrate abnormal function of the cochlear nerve despite typical function of sensory cells. Because the human inner ear cannot be visualized during life, histopathological analysis of autopsy specimens is critical to understanding the cellular mechanisms underlying this pathology. Here we present statistical models of severe primary neuronal degeneration and its relationship to pure tone audiometric thresholds and word recognition scores in comparison to age-matched control patients, spanning every decade of life. Analysis of 30 ears from 23 patients shows that severe neuronal loss correlates with elevated audiometric thresholds and poor word recognition. For each ten percent increase in total neuronal loss, average thresholds across patients at each audiometric test frequency increase by 6.0 dB hearing level (HL). As neuronal loss increases, threshold elevation proceeds more rapidly in low audiometric test frequencies than in high frequencies. Pure tone average closely agrees with word recognition scores in the case of severe neural pathology. Histopathologic study of the human inner ear continues to emphasize the need for non- or minimally invasive clinical tools capable of establishing cellular-level diagnoses.

  View details for DOI 10.1038/s41598-017-04899-9

  View details for Web of Science ID 000404970900046

  View details for PubMedID 28687782

  View details for PubMedCentralID PMC5501826

• Skin Pigmentation and Risk of Hearing Loss in Women AMERICAN JOURNAL OF EPIDEMIOLOGY Lin, B. M., Li, W., Curhan, S. G., Stankovic, K. M., Qureshi, A. A., Curhan, G. C. 2017; 186 (1): 1-10

  Abstract

  Black individuals have a lower risk of hearing loss than do whites, possibly because of differences in cochlear melanocytes. Previous studies have suggested that darker-skinned individuals tend to have more inner ear melanin, and cochlear melanocytes are important in generating the endocochlear potential. We investigated the relationship between self-reported hearing loss and skin pigmentation by using hair color, skin tanning ability, and skin reaction to prolonged sun exposure as surrogate measures of pigmentation among 49,323 white women in the Nurses' Health Study. Cox proportional hazards regression models were used to adjust for potential confounders. During 1,190,170 person-years of follow-up (1982-2012), there was no association between risk of hearing loss and hair color (for black hair vs. red or blonde hair, multivariable-adjusted relative risk (RR) = 0.99, 95% confidence interval (CI): 0.90, 1.09), skin tanning ability (for dark tan vs. no tan, multivariable-adjusted RR = 0.98, 95% CI: 0.92, 1.05), skin reaction to prolonged sun exposure (for painful burn with blisters vs. practically no reaction, multivariable-adjusted RR = 1.01, 95% CI: 0.93, 1.08), or Fitzpatrick skin phototype (for type IV vs. type I, multivariable-adjusted RR = 0.99, 95% CI: 0.92, 1.05). In our cohort of white women, surrogates for skin pigmentation were not associated with risk of hearing loss.

  View details for DOI 10.1093/aje/kwx024

  View details for Web of Science ID 000405477300001

  View details for PubMedID 28525626

  View details for PubMedCentralID PMC5860009

• Cochlear Implantation: Vast Unmet Need to Address Deafness Globally OTOLOGY & NEUROTOLOGY Sahin, M., Sagers, J. E., Stankovic, K. M. 2017; 38 (6): 786-787

  View details for DOI 10.1097/MAO.0000000000001416

  View details for Web of Science ID 000403225200020

  View details for PubMedID 28437361

  View details for PubMedCentralID PMC5864459

• Neonatal Murine Cochlear Explant Technique as an In Vitro Screening Tool in Hearing Research. Journal of visualized experiments : JoVE Landegger, L. D., Dilwali, S., Stankovic, K. M. 2017

  Abstract

  While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a condition that typically involves damage to or loss of the delicate mechanosensory structures of the inner ear. Sophisticated in vitro and ex vivo assays have emerged in recent years, enabling the screening of an increasing number of potentially therapeutic compounds while minimizing resources and accelerating efforts to develop cures for SNHL. Though homogenous cultures of certain cell types continue to play an important role in current research, many scientists now rely on more complex organotypic cultures of murine inner ears, also known as cochlear explants. The preservation of organized cellular structures within the inner ear facilitates the in situ evaluation of various components of the cochlear infrastructure, including inner and outer hair cells, spiral ganglion neurons, neurites, and supporting cells. Here we present the preparation, culture, treatment, and immunostaining of neonatal murine cochlear explants. The careful preparation of these explants facilitates the identification of mechanisms that contribute to SNHL and constitutes a valuable tool for the hearing research community.

  View details for DOI 10.3791/55704

  View details for PubMedID 28654047

  View details for PubMedCentralID PMC5608349

• A Unified Methodological Framework for Vestibular Schwannoma Research JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Landegger, L. D., Sagers, J. E., Dilwali, S., Fujita, T., Sahin, M. I., Stankovic, K. M. 2017

  Abstract

  Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these tumors cause sensorineural hearing loss in up to 95% of affected individuals, the molecular mechanisms underlying this hearing loss remain elusive. This article outlines the steps established in our laboratory to facilitate the collection and processing of various primary human tissue samples for downstream research applications integral to the study of vestibular schwannomas. Specifically, this work describes a unified methodological framework for the collection, processing, and culture of Schwann and schwannoma cells from surgical samples. This is integrated with parallel processing steps now considered essential for current research: the collection of tumor and nerve secretions, the preservation of RNA and the extraction of protein from collected tissues, the fixation of tissue for the preparation of sections, and the exposure of primary human cells to adeno-associated viruses for application to gene therapy. Additionally, this work highlights the translabyrinthine surgical approach to collect this tumor as a unique opportunity to obtain human sensory epithelium from the inner ear and perilymph. Tips to improve experimental quality are provided and common pitfalls highlighted.

  View details for DOI 10.3791/55827

  View details for Web of Science ID 000415751100017

  View details for PubMedID 28654042

  View details for PubMedCentralID PMC5608471

• A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear NATURE BIOTECHNOLOGY Landegger, L. D., Pan, B., Askew, C., Wassmer, S. J., Gluck, S. D., Galvin, A., Taylor, R., Forge, A., Stankovic, K. M., Holt, J. R., Vandenberghe, L. H. 2017; 35 (3): 280-+

  Abstract

  Efforts to develop gene therapies for hearing loss have been hampered by the lack of safe, efficient, and clinically relevant delivery modalities. Here we demonstrate the safety and efficiency of Anc80L65, a rationally designed synthetic vector, for transgene delivery to the mouse cochlea. Ex vivo transduction of mouse organotypic explants identified Anc80L65 from a set of other adeno-associated virus (AAV) vectors as a potent vector for the cochlear cell targets. Round window membrane injection resulted in highly efficient transduction of inner and outer hair cells in mice, a substantial improvement over conventional AAV vectors. Anc80L65 round window injection was well tolerated, as indicated by sensory cell function, hearing and vestibular function, and immunologic parameters. The ability of Anc80L65 to target outer hair cells at high rates, a requirement for restoration of complex auditory function, may enable future gene therapies for hearing and balance disorders.

  View details for DOI 10.1038/nbt.3781

  View details for Web of Science ID 000395924600027

  View details for PubMedID 28165475

  View details for PubMedCentralID PMC5340646

• Prospective Study of Gastroesophageal Reflux, Use of Proton Pump Inhibitors and H2-Receptor Antagonists, and Risk of Hearing Loss EAR AND HEARING Lin, B. M., Curhan, S. G., Wang, M., Jacobson, B. C., Eavey, R., Stankovic, K. M., Curhan, G. C. 2017; 38 (1): 21-27

  Abstract

  Gastroesophageal reflux disease (GERD) is common and often treated with proton pump inhibitors (PPIs) or H2-receptor antagonists (H2-RAs). GERD has been associated with exposure of the middle ear to gastric contents, which could cause hearing loss. Treatment of GERD with PPIs and H2-RAs may decrease exposure of the middle ear to gastric acid and decrease the risk of hearing loss. We prospectively investigated the relation between GERD, use of PPIs and H2-RAs, and the risk of hearing loss in 54,883 women in Nurses' Health Study II.Eligible participants, aged 41 to 58 years in 2005, provided information on medication use and GERD symptoms in 2005, answered the question on hearing loss in 2009 or in 2013, and did not report hearing loss starting before the date of onset of GERD symptoms or medication use. The primary outcome was self-reported hearing loss. Cox proportional hazards regression was used to adjust for potential confounders.During 361,872 person-years of follow-up, 9842 new cases of hearing loss were reported. Compared with no GERD symptoms, higher frequency of GERD symptoms was associated with higher risk of hearing loss (multivariable adjusted relative risks: <1 time/month 1.04 [0.97, 1.11], several times/week 1.17 [1.09, 1.25], daily 1.33 [1.19, 1.49]; p value for trend <0.001). After accounting for GERD symptoms, neither PPI nor H2-RA use was associated with the risk of hearing loss.GERD symptoms are associated with higher risk of hearing loss in women, but use of PPIs and H2-RAs are not independently associated with the risk.

  View details for DOI 10.1097/AUD.0000000000000347

  View details for Web of Science ID 000391630200010

  View details for PubMedID 27556519

  View details for PubMedCentralID PMC5161691

• Duration of Analgesic Use and Risk of Hearing Loss in Women AMERICAN JOURNAL OF EPIDEMIOLOGY Lin, B. M., Curhan, S. G., Wang, M., Eavey, R., Stankovic, K. M., Curhan, G. C. 2017; 185 (1): 40-47

  Abstract

  Aspirin, nonsteroidal antiinflammatory drugs (NSAID), and acetaminophen are commonly used. Frequent use of analgesics has been associated with a higher risk of hearing loss. However, the association between duration of analgesic use and the risk of hearing loss is unclear. We investigated the relationship between duration of analgesic use and self-reported hearing loss among 55,850 women in the Nurses' Health Study. Cox proportional hazards regression was used to adjust for potential confounders. During 873,376 person-years of follow-up (1990-2012), longer durations of NSAID use (for >6 years of use compared with <1 year, multivariable-adjusted relative risk = 1.10, 95% confidence interval: 1.06, 1.15; P for trend < 0.001) and acetaminophen use (for >6 years of use compared with <1 year, multivariable-adjusted relative risk = 1.09, 95% confidence interval: 1.04, 1.14; P for trend < 0.001) were associated with higher risks of hearing loss. Duration of aspirin use was not associated with hearing loss (for >6 years of use compared with <1 year, multivariable-adjusted relative risk = 1.01, 95% confidence interval: 0.97, 1.05; P for trend = 0.35). In this cohort of women, longer durations of NSAID and acetaminophen use were associated with slightly higher risks of hearing loss, but duration of aspirin use was not. Considering the high prevalence of analgesic use, this may be an important modifiable contributor to hearing loss.

  View details for DOI 10.1093/aje/kww154

  View details for Web of Science ID 000397232100006

  View details for PubMedID 27974293

  View details for PubMedCentralID PMC5209586

• Hepatocyte Nuclear Factor-4 Alpha in Noise-Induced Cochlear Neuropathy DEVELOPMENTAL NEUROBIOLOGY Groth, J., Kao, S., Briet, M. C., Stankovic, K. M. 2016; 76 (12): 1374-1386

  Abstract

  Noise-induced hearing loss (NIHL) is a problem of profound clinical significance and growing magnitude. Alarmingly, even moderate noise levels, previously assumed to cause only temporary shifts in auditory thresholds ("temporary" NIHL), are now known to cause cochlear synaptopathy and subsequent neuropathy. To uncover molecular mechanisms of this neuropathy, a network analysis of genes reported to have significantly altered expression after temporary threshold shift-inducing noise exposure was performed. The transcription factor Hepatocyte Nuclear Factor-4 alpha (HNF4α), which had not previously been studied in the context of cochlear response to noise, was identified as a hub of a top-ranking network. Hnf4α expression and localization using quantitative RT-PCR and in situ hybridization, respectively, were described in adolescent and adult mice exposed to neuropathic noise levels in adolescence. Isoforms α3 and α12 in the cochlea were also identified. At every age examined, Hnf4α mRNA expression in the cochlear apex was similar to expression in the base. Hnf4α expression was evident in select cochlear cells, including spiral ganglion neurons (SGNs) and hair cells, and was significantly upregulated from 6 to 70 weeks of age, especially in SGNs. This age-related Hnf4α upregulation was inhibited by neuropathic noise exposure in adolescence. Hnf4α silencing with shRNA transfection into auditory neuroblast cells (VOT-33) reduced cell viability, as measured with the MTT assay, suggesting that Hnf4α may be involved in SGN survival. Our results motivate future studies of HNF4α in cochlear pathophysiology, especially because HNF4α mutations and polymorphisms are associated with human diseases that may include hearing loss. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 76: 1374-1386, 2016.

  View details for DOI 10.1002/dneu.22399

  View details for Web of Science ID 000390008700006

  View details for PubMedID 27112738

• The Impact of Angiotensin-Modulating Antihypertensives on Time Interval to Revision Surgery for Nasal Polyps OTOLARYNGOLOGY-HEAD AND NECK SURGERY Brook, C. D., Maxfield, A. Z., Stankovic, K., Metson, R. B. 2016; 155 (6): 1046-1052

  Abstract

  Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) have been shown to suppress expression of periostin, a matricellular protein that is markedly elevated in nasal polyp tissue. The purpose of this study was to determine whether use of these antihypertensive agents affects the time to revision sinus surgery in patients with polyp regrowth.Case series with chart review.Academic medical center.Records were reviewed for 330 patients who underwent ≥2 operations for chronic sinusitis with nasal polyps from April 1987 through August 2015. The time between surgical interventions was compared with patient demographics and clinical characteristics, including use of ACEIs and ARBs.Sixty patients were taking ACEIs or ARBs during the study period, of which 32 had concurrent asthma. The mean interval between polyp operations was 61.0 ± 45.2 months (range, 2-228.6 months). Among patients with asthma (n = 197), the mean time to revision surgery was prolonged by >2 years for those taking ACEIs or ARBs (81.0 vs 54.5 months, P = .006). A similar impact on time to revision surgery was not observed for nonasthmatics taking these medications (61.0 vs 65.2 months, P = .655).Use of ACEIs and ARBs is associated with an increased time to revision sinus surgery among patients with concurrent nasal polyps and asthma. A possible mechanism of this observed effect is suppression of periostin expression through inhibition of the angiotensin pathway.

  View details for DOI 10.1177/0194599816663924

  View details for Web of Science ID 000389146500021

  View details for PubMedID 27554516

• Sulforaphane, a natural component of broccoli, inhibits vestibular schwannoma growth in vitro and in vivo SCIENTIFIC REPORTS Kim, B., Fujita, T., Stankovic, K. M., Welling, D., Moon, I., Choi, J., Yun, J., Kang, J., Lee, J. 2016; 6: 36215

  Abstract

  Vestibular schwannoma (VS) is an intracranial tumor that causes significant morbidity, including hearing loss, tinnitus, dizziness, and possibly even death from brainstem compression. However, FDA-approved pharmacologic treatments for VS do not exist. Sulforaphane (SFN) is a naturally occurring isothiocyanate found in cruciferous vegetables, such as broccoli, with potent chemoprotective effects in several cell types. Our objective was to determine whether SFN is effective against VS in vitro and in vivo. Human primary VS cells, HEI-193 schwannoma cells, and SC4 Nf2-/- Schwann cells were used to investigate the inhibitory effects of SFN in vitro. Cell proliferation was assessed by bromodeoxyuridine (BrdU) incorporation, and cell viability and metabolic activity was calculated by MTT assay. Apoptosis was measured by flow cytometry, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and Western blot for cleaved caspases. A mouse model with a murine schwannoma allograft was also used to examine the antitumor activity of SFN. SFN exhibited significant antiproliferative activity in schwannoma cells in vitro, via the inhibition of HDAC activity and the activation of ERK. SFN treatment induced apoptosis and cell cycle arrest at the G2/M phase. SFN also significantly inhibited schwannoma growth in vivo. Our preclinical studies motivate a future prospective clinical study of SFN for the treatment of VS.

  View details for DOI 10.1038/srep36215

  View details for Web of Science ID 000387137200001

  View details for PubMedID 27805058

  View details for PubMedCentralID PMC5090244

• Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells NEURO-ONCOLOGY Soares, V. R., Atai, N. A., Fujita, T., Dilwali, S., Sivaraman, S., Landegger, L. D., Hochberg, F. H., Oliveira, C. C., Bahmad, F., Breakefield, X. O., Stankovic, K. M. 2016; 18 (11): 1498-1507

  Abstract

  Vestibular schwannoma (VS) is a tumor of the vestibular nerve that transmits balance information from the inner ear to the brain. Sensorineural hearing loss occurs in 95% of patients with these tumors, but the cause of this loss is not well understood. We posit a role of VS-secreted extracellular vesicles (EVs) as a major contributing factor in cochlear nerve damage.Using differential centrifugation, we isolated EVs from VS cell line HEI-193 and primary cultured human VS cells from patients with good hearing or poor hearing. The EVs were characterized using a Nanosight device and transmission electron microscopy and by extracting their RNA content. The EVs' effects on cultured murine spiral ganglion cells and organotypic cochlear cultures were studied using a transwell dual-culture system and by direct labeling of EVs with PKH-67 dye. EV-induced changes in cochlear cells were quantified using confocal immunohistochemistry. Transfection of VS cells with a green fluorescent protein-containing plasmid was confirmed with reverse transcription PCR.Human VS cells, from patients with poor hearing, produced EVs that could damage both cultured murine cochlear sensory cells and neurons. In contrast, EVs derived from VS cells from patients with good hearing did not damage the cultured cochlear cells.This is the first report on EVs derived from VSs and on the capacity of EVs from VSs from patients with hearing loss to selectively damage cochlear cells, thereby identifying a potential novel mechanism of VS-associated sensorineural hearing loss.

  View details for DOI 10.1093/neuonc/now099

  View details for Web of Science ID 000387330300006

  View details for PubMedID 27194145

  View details for PubMedCentralID PMC5063517

• Correlation Between Aspirin Intake and Reduced Growth of Human Vestibular Schwannoma: Volumetric Analysis OTOLOGY & NEUROTOLOGY Kandathil, C. K., Cunnane, M. E., McKenna, M. J., Curtin, H. D., Stankovic, K. M. 2016; 37 (9): 1428-1434

  Abstract

  To determine whether people with sporadic vestibular schwannoma (VS) who take aspirin for unrelated medical reasons exhibit less tumor growth than nonaspirin users. We previously demonstrated the efficacy of salicylates in inhibiting VS growth in vitro, corroborating the results of our retrospective clinical study, which found halted VS growth (based on linear tumor measurements) in aspirin users. The current study evaluates this association using more accurate tumor volumetric measurements, and quantifies the degree of frequency-specific, VS-induced hearing loss.Retrospective analysis.Tertiary care hospital.Diagnosed with VS between 1980 and 2012, followed by serial magnetic resonance imaging for at least 1 year.Patient history of aspirin intake; change in VS volume over time of observation; frequency-specific, VS-induced audiometric threshold shifts.Of the 347 patients followed by serial magnetic resonance imaging scans, 86 had sequential scans available for 3D-segmented volumetric analysis for up to 11 years of follow-up (median 53 mo). Twenty-five (29%) had documented history of aspirin intake; 8 (32%) of these demonstrated VS growth. Of the 61 (71%) nonusers, 36 (59%) demonstrated growth. A significant inverse association was found among aspirin users and VS growth: odds ratio 0.32, 95% confidence interval 0.11 to 0.91. VS-induced audiometric thresholds shifts were larger above than below 2000 Hz.Our volumetric analysis of VS growth reaffirms the results of our linear analysis and suggests that aspirin may inhibit VS growth. The audiometric findings are consistent with the previously reported VS-induced predominantly high-frequency sensorineural hearing loss.

  View details for DOI 10.1097/MAO.0000000000001180

  View details for Web of Science ID 000384464300051

  View details for PubMedID 27631829

• Micro-optical coherence tomography of the mammalian cochlea SCIENTIFIC REPORTS Iyer, J. S., Batts, S. A., Chu, K. K., Sahin, M. I., Leung, H., Tearney, G. J., Stankovic, K. M. 2016; 6: 33288

  Abstract

  The mammalian cochlea has historically resisted attempts at high-resolution, non-invasive imaging due to its small size, complex three-dimensional structure, and embedded location within the temporal bone. As a result, little is known about the relationship between an individual's cochlear pathology and hearing function, and otologists must rely on physiological testing and imaging methods that offer limited resolution to obtain information about the inner ear prior to performing surgery. Micro-optical coherence tomography (μOCT) is a non-invasive, low-coherence interferometric imaging technique capable of resolving cellular-level anatomic structures. To determine whether μOCT is capable of resolving mammalian intracochlear anatomy, fixed guinea pig inner ears were imaged as whole temporal bones with cochlea in situ. Anatomical structures such as the tunnel of Corti, space of Nuel, modiolus, scalae, and cell groupings were visualized, in addition to individual cell types such as neuronal fibers, hair cells, and supporting cells. Visualization of these structures, via volumetrically-reconstructed image stacks and endoscopic perspective videos, represents an improvement over previous efforts using conventional OCT. These are the first μOCT images of mammalian cochlear anatomy, and they demonstrate μOCT's potential utility as an imaging tool in otology research.

  View details for DOI 10.1038/srep33288

  View details for Web of Science ID 000383331800001

  View details for PubMedID 27633610

  View details for PubMedCentralID PMC5025881

• Surgical Anatomy of the Human Round Window Region: Implication for Cochlear Endoscopy Through the External Auditory Canal. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology Fujita, T., Shin, J. E., Cunnane, M., Fujita, K., Henein, S., Psaltis, D., Stankovic, K. M. 2016; 37 (8): 1189-94

  Abstract

  To enable development of an endoscope for cellular-level optical imaging of the inner ear.A prospective study of 50 cadaveric human temporal bones to define detailed surgical anatomy of the round window (RW) region and the range of angles necessary to reach the RW membrane perpendicularly via the external ear canal.The transcanal angle to the RW membrane was surgically measured in 3D intact specimens, and correlated with the angle calculated from temporal bone computed tomography (CT) scans of the same specimens obtained before and after measurements in situ.Surgically measured transcanal angles to the RW membrane correlated well with the radiographically measured angles. The angles ranged from 110 to 127 degrees, with the median of 115 degrees and the middle 50% ranging from 109 to 119 degrees. Four temporal bones were excluded because of pathology. The opening of the RW niche was located posteriorly in six bones (13%), inferiorly in 18 bones (39%), and postero-inferiorly in 22 bones (48%). The angles were not statistically different among the three orientations of the RW niche.By correlating measurement from cadaveric human temporal bones and their CT scans, we defined key parameters necessary for designing an endoscope for intracochlear imaging using a minimally invasive approach through the external auditory canal. The excellent correlation between the measurement on the CT scan and the actual shape of the probe that was able to reach the RW through the ear canal enables selection of the probe using the CT data.

  View details for DOI 10.1097/MAO.0000000000001074

  View details for PubMedID 27228017

• Human audiometric thresholds do not predict specific cellular damage in the inner ear HEARING RESEARCH Landegger, L. D., Psaltis, D., Stankovic, K. M. 2016; 335: 83-93

  Abstract

  As otology enters the field of gene therapy and human studies commence, the question arises whether audiograms - the current gold standard for the evaluation of hearing function - can consistently predict cellular damage within the human inner ear and thus should be used to define inclusion criteria for trials. Current assumptions rely on the analysis of small groups of human temporal bones post mortem or from psychophysical identification of cochlear "dead regions" in vivo, but a comprehensive study assessing the correlation between audiometric thresholds and cellular damage within the cochlea is lacking.A total of 131 human temporal bones from 85 adult individuals (ages 19-92 years, median 69 years) with sensorineural hearing loss due to various etiologies were analyzed. Cytocochleograms - which quantify loss of hair cells, neurons, and strial atrophy along the length of the cochlea - were compared with subjects' latest available audiometric tests prior to death (time range 5 h-22 years, median 24 months). The Greenwood function and the equivalent rectangular bandwidth were used to infer, from cytocochleograms, cochlear locations corresponding to frequencies tested in clinical audiograms. Correlation between audiometric thresholds at clinically tested frequencies and cell type-specific damage in those frequency regions was examined by calculating Spearman's correlation coefficients.Similar audiometric profiles reflected widely different cellular damage in the cochlea. In our diverse group of patients, audiometric thresholds tended to be more influenced by hair cell loss than by neuronal loss or strial atrophy. Spearman's correlation coefficient across frequencies was at most 0.7 and often below 0.5, with 1.0 indicating perfect correlation.Audiometric thresholds do not predict specific cellular damage in the human inner ear. Our study highlights the need for better non- or minimally-invasive tools, such as cochlear endoscopy, to establish cellular-level diagnosis and thereby guide therapy and monitor response to treatment.

  View details for DOI 10.1016/j.heares.2016.02.018

  View details for Web of Science ID 000376713900011

  View details for PubMedID 26924453

  View details for PubMedCentralID PMC5970796

• Hypertension, Diuretic Use, and Risk of Hearing Loss AMERICAN JOURNAL OF MEDICINE Lin, B. M., Curhan, S. G., Wang, M., Eavey, R., Stankovic, K. M., Curhan, G. C. 2016; 129 (4): 416-422

  Abstract

  Hearing loss is highly prevalent among adults in the United States. Hypertension also is common and often treated with diuretics. Hypertension may increase the risk of hearing loss by decreasing vascular supply to the stria vascularis. Use of thiazides has been anecdotally associated with hearing loss. In small studies, furosemide use has been associated with hearing loss that is usually reversible, but can be permanent. We investigated the relation among hypertension, diuretic use, and hearing loss in a prospective cohort of 54,721 women in the Nurses' Health Study I, 1994 to 2012.Eligible participants included 54,721 female nurses aged 48 to 73 years in 1994 who provided information on thiazide diuretic and furosemide use in 1994, answered the question on hearing loss over their lifetime in 2012, and did not report hearing loss with date of onset before date of onset of hypertension diagnosis or medication use. The outcome was self-reported hearing loss. Cox proportional hazards regression was used to adjust for potential confounders.During 774,096 person-years of follow-up, 19,296 cases of hearing loss were reported (incidence rate, 25 cases per 1000 person-years). At baseline in 1994, the mean age was 57.9 years and mean body mass index was 26.3 kg/m(2). Some 30.8% of participants had a history of hypertension. History of hypertension was independently associated with a modestly higher risk of hearing loss (multivariable adjusted relative risk, 1.04 [1.01-1.07]). Among women with a history of hypertension, neither thiazide diuretic (multivariable adjusted relative risk, 1.07 [0.99-1.16]) nor furosemide use (multivariable adjusted relative risk, 0.91 [0.75-1.09]) was significantly associated with risk of hearing loss when compared with women not taking antihypertensive medications. There was no significant effect modification by age.History of hypertension was associated with a small increased risk of hearing loss. Thiazide diuretic use and furosemide use were not associated with risk of hearing loss among women with a history of hypertension.

  View details for DOI 10.1016/j.amjmed.2015.11.014

  View details for Web of Science ID 000373486500028

  View details for PubMedID 26656761

  View details for PubMedCentralID PMC4792671

• Activation of TRAIL-DR5 pathway promotes sensorineural degeneration in the inner ear AGING CELL Kao, S., Soares, V. R., Kristiansen, A. G., Stankovic, K. M. 2016; 15 (2): 301-308

  Abstract

  Tumor necrosis factor (TNF) family cytokines are important mediators of inflammation. Elevated levels of serum TNF-α are associated with human sensorineural hearing loss via poorly understood mechanisms. We demonstrate, for the first time, expression of TNF-related apoptosis-inducing ligand (TRAIL) and its signaling death receptor 5 (DR5) in the murine inner ear and show that exogenous TRAIL can trigger hair cell and neuronal degeneration, which can be partly prevented with DR5-blocking antibodies.

  View details for DOI 10.1111/acel.12437

  View details for Web of Science ID 000372880500011

  View details for PubMedID 26791792

  View details for PubMedCentralID PMC4783338

• Revealing Hearing Loss: A Survey of How People Verbally Disclose Their Hearing Loss EAR AND HEARING West, J. S., Low, J. M., Stankovic, K. M. 2016; 37 (2): 194-205

  Abstract

  Hearing loss is the most common sensory deficit and congenital anomaly, yet the decision-making processes involved in disclosing hearing loss have been little studied. To address this issue, we have explored the phrases that adults with hearing loss use to disclose their hearing loss.Since self-disclosure research has not focused on hearing loss-specific issues, we created a 15-question survey about verbally disclosing hearing loss. English speaking adults (>18 years old) with hearing loss of any etiology were recruited from otology clinics in a major referral hospital. Three hundred and thirty-seven participants completed the survey instrument. Participants' phrase(s) used to tell people they have hearing loss were compared across objective characteristics (age; sex; type, degree, and laterality of hearing loss; word recognition scores) and self-reported characteristics (degree of hearing loss; age of onset and years lived with hearing loss; use of technology; hearing handicap score).Participants' responses revealed three strategies to address hearing loss: Multipurpose disclosure (phrases that disclose hearing loss and provide information to facilitate communication), Basic disclosure (phrases that disclose hearing loss through the term, a label, or details about the condition), or nondisclosure (phrases that do not disclose hearing loss). Variables were compared between patients who used and who did not use each disclosure strategy using χ or Wilcoxon rank sum tests. Multipurpose disclosers were mostly female (p = 0.002); had experienced reactions of help, support, and accommodation after disclosing (p = 0.008); and had experienced reactions of being overly helpful after disclosing (p=0.039). Basic disclosers were predominantly male (p = 0.004); reported feeling somewhat more comfortable disclosing their hearing loss over time (p = 0.009); had not experienced reactions of being treated unfairly or discriminated against (p = 0.021); and were diagnosed with mixed hearing loss (p = 0.004). Nondisclosers tended not to disclose in a group setting (p = 0.002) and were diagnosed with bilateral hearing loss (p = 0.005). In addition, all of the variables were examined to build logistic regression models to predict the use of each disclosure strategy.Our results reveal three simple strategies for verbally addressing hearing loss that can be used in a variety of contexts. We recommend educating people with hearing loss about these strategies-this could improve the experience of disclosing hearing loss, and could educate society at large about how to interact with those who have a hearing loss.

  View details for DOI 10.1097/AUD.0000000000000238

  View details for Web of Science ID 000371748300008

  View details for PubMedID 26513208

  View details for PubMedCentralID PMC6721611

• Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage SCIENTIFIC REPORTS Dilwali, S., Landegger, L. D., Soares, V. R., Deschler, D. G., Stankovic, K. M. 2015; 5: 18599

  Abstract

  Vestibular schwannomas (VSs) are the most common tumours of the cerebellopontine angle. Ninety-five percent of people with VS present with sensorineural hearing loss (SNHL); the mechanism of this SNHL is currently unknown. To establish the first model to study the role of VS-secreted factors in causing SNHL, murine cochlear explant cultures were treated with human tumour secretions from thirteen different unilateral, sporadic VSs of subjects demonstrating varied degrees of ipsilateral SNHL. The extent of cochlear explant damage due to secretion application roughly correlated with the subjects' degree of SNHL. Secretions from tumours associated with most substantial SNHL resulted in most significant hair cell loss and neuronal fibre disorganization. Secretions from VSs associated with good hearing or from healthy human nerves led to either no effect or solely fibre disorganization. Our results are the first to demonstrate that secreted factors from VSs can lead to cochlear damage. Further, we identified tumour necrosis factor alpha (TNFα) as an ototoxic molecule and fibroblast growth factor 2 (FGF2) as an otoprotective molecule in VS secretions. Antibody-mediated TNFα neutralization in VS secretions partially prevented hair cell loss due to the secretions. Taken together, we have identified a new mechanism responsible for SNHL due to VSs.

  View details for DOI 10.1038/srep18599

  View details for Web of Science ID 000367032600001

  View details for PubMedID 26690506

  View details for PubMedCentralID PMC4686978

• CASE RECORDS of the MASSACHUSETTS GENERAL HOSPITAL. Case 36-2015. A 27-Year-Old Woman with a Lesion of the Ear Canal. The New England journal of medicine Stankovic, K. M., Tan, O. T., Sadow, P. M. 2015; 373 (21): 2070-7

  View details for DOI 10.1056/NEJMcpc1410941

  View details for PubMedID 26581000

• Carotenoids, vitamin A, vitamin C, vitamin E, and folate and risk of self-reported hearing loss in women. The American journal of clinical nutrition Curhan, S. G., Stankovic, K. M., Eavey, R. D., Wang, M., Stampfer, M. J., Curhan, G. C. 2015; 102 (5): 1167-75

  Abstract

  Higher intake of certain vitamins may protect against cochlear damage from vascular compromise and oxidative stress, thereby reducing risk of acquired hearing loss, but data are limited.We prospectively examined the relation between carotenoids, vitamin A, vitamin C, vitamin E, and folate intake and risk of self-reported hearing loss in women.This prospective cohort study followed 65,521 women in the Nurses' Health Study II from 1991 to 2009. Baseline and updated information obtained from validated biennial questionnaires was used in Cox proportional hazards regression models to examine independent associations between nutrient intake and self-reported hearing loss.After 1,084,598 person-years of follow-up, 12,789 cases of incident hearing loss were reported. After multivariable adjustment, we observed modest but statistically significant inverse associations between higher intake of β-carotene and β-cryptoxanthin and risk of hearing loss. In comparison with women in the lowest quintile of intake, the multivariable-adjusted RR of hearing loss among women in the highest quintile was 0.88 (95% CI: 0.81, 0.94; P-trend < 0.001) for β-carotene and 0.90 (95% CI: 0.84, 0.96; P-trend < 0.001) for β-cryptoxanthin. In comparison with women with folate intake 200-399 μg/d, very low folate intake (<200 μg/d) was associated with higher risk (RR: 1.19; 95% CI: 1.01, 1.41), and higher intake tended to be associated with lower risk (P-trend = 0.04). No significant associations were observed for intakes of other carotenoids or vitamin A. Higher vitamin C intake was associated with higher risk; in comparison with women with intake <75 mg/d, the RR among women with vitamin C intake ≥1000 mg/d (mainly supplemental) was 1.22 (95% CI: 1.06, 1.42; P-trend = 0.02). There was no significant trend between intake of vitamin E intake and risk.Higher intakes of β-carotene, β-cryptoxanthin, and folate, whether total or from diet, are associated with lower risk of hearing loss, whereas higher vitamin C intake is associated with higher risk.

  View details for DOI 10.3945/ajcn.115.109314

  View details for PubMedID 26354537

  View details for PubMedCentralID PMC4625586

• Preclinical validation of anti-nuclear factor-kappa B therapy to inhibit human vestibular schwannoma growth MOLECULAR ONCOLOGY Dilwali, S., Briet, M. C., Kao, S., Fujita, T., Landegger, L. D., Platt, M. P., Stankovic, K. M. 2015; 9 (7): 1359-1370

  Abstract

  Vestibular schwannomas (VSs), the most common tumors of the cerebellopontine angle, arise from Schwann cells lining the vestibular nerve. Pharmacotherapies against VS are almost non-existent. Although the therapeutic inhibition of inflammatory modulators has been established for other neoplasms, it has not been explored in VS. A bioinformatic network analysis of all genes reported to be differentially expressed in human VS revealed a pro-inflammatory transcription factor nuclear factor-kappa B (NF-κB) as a central molecule in VS pathobiology. Assessed at the transcriptional and translational level, canonical NF-κB complex was aberrantly activated in human VS and derived VS cultures in comparison to control nerves and Schwann cells, respectively. Cultured primary VS cells and VS-derived human cell line HEI-193 were treated with specific NF-κB siRNAs, experimental NF-κB inhibitor BAY11-7082 (BAY11) and clinically relevant NF-κB inhibitor curcumin. Healthy human control Schwann cells from the great auricular nerve were also treated with BAY11 and curcumin to assess toxicity. All three treatments significantly reduced proliferation in primary VS cultures and HEI-193 cells, with siRNA, 5 μM BAY11 and 50 μM curcumin reducing average proliferation (±standard error of mean) to 62.33% ± 10.59%, 14.3 ± 9.7%, and 23.0 ± 20.9% of control primary VS cells, respectively. These treatments also induced substantial cell death. Curcumin, unlike BAY11, also affected primary Schwann cells. This work highlights NF-κB as a key modulator in VS cell proliferation and survival and demonstrates therapeutic efficacy of directly targeting NF-κB in VS.

  View details for DOI 10.1016/j.molonc.2015.03.009

  View details for Web of Science ID 000359884800011

  View details for PubMedID 25891780

  View details for PubMedCentralID PMC4523465

• Transactivation of human osteoprotegerin promoter by GATA-3 SCIENTIFIC REPORTS Kao, S., Stankovic, K. M. 2015; 5: 12479

  Abstract

  Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations in OPG are involved in a variety of human diseases. We have shown that cochlear spiral ganglion cells secrete OPG at high levels and lack of OPG causes sensorineural hearing loss in addition to the previously described conductive hearing loss. In order to study the regulation of OPG expression, we conducted a database search on regulatory elements in the promoter region of the OPG gene, and identified two potential GATA-3 binding sites. Using luciferase assays and site directed mutagenesis, we demonstrate that these two elements are GATA-3 responsive and support GATA-3 transactivation in human HEK and HeLa cells. The expression of wild type GATA-3 activated OPG mRNA and protein expression, while the expression of a dominant negative mutant of GATA-3 or a GATA-3 shRNA construct reduced OPG mRNA and protein levels. GATA-3 deficient cells generated by expressing a GATA-3 shRNA construct were sensitive to apoptosis induced by etoposide and TNF-α. This apoptotic effect could be partly prevented by the co-treatment with exogenous OPG. Our results suggest new approaches to rescue diseases due to GATA-3 deficiency - such as in hypoparathyroidism, sensorineural deafness, and renal (HDR) syndrome - by OPG therapy.

  View details for DOI 10.1038/srep12479

  View details for Web of Science ID 000358586100001

  View details for PubMedID 26216189

  View details for PubMedCentralID PMC4516985

• Nonsteroidal anti-inflammatory medications are cytostatic against human vestibular schwannomas. Translational research : the journal of laboratory and clinical medicine Dilwali, S., Kao, S. Y., Fujita, T., Landegger, L. D., Stankovic, K. M. 2015; 166 (1): 1-11

  Abstract

  Vestibular schwannomas (VSs) are the most common tumors of the cerebellopontine angle. Significant clinical need exists for pharmacotherapies against VSs. Motivated by previous findings that immunohistochemical expression of cyclooxygenase 2 (COX-2) correlates with VS growth rate, we investigated the role of COX-2 in VSs and tested COX-2 inhibiting salicylates against VSs. COX-2 was found to be aberrantly expressed in human VS and primary human VS cells in comparison with control human nerve specimens and primary Schwann cells (SCs), respectively. Furthermore, levels of prostaglandin E2, the downstream enzymatic product of COX-2, were correlated with primary VS culture proliferation rate. Because COX-2 inhibiting salicylates such as aspirin are well tolerated and frequently clinically used, we assessed their repurposing for VS. Changes in proliferation, cell death, and cell viability were analyzed in primary VS cultures treated with aspirin, sodium salicylate, or 5-aminosalicylic acid. These drugs neither increased VS cell death nor affected healthy SCs. The cytostatic effect of aspirin in vitro was in concurrence with our previous clinical finding that patients with VS taking aspirin demonstrate reduced tumor growth. Overall, this work suggests that COX-2 is a key modulator in VS cell proliferation and survival and highlights salicylates as promising pharmacotherapies against VS.

  View details for DOI 10.1016/j.trsl.2014.12.007

  View details for PubMedID 25616959

  View details for PubMedCentralID PMC4458444

• Immediate and Delayed Cochlear Neuropathy after Noise Exposure in Pubescent Mice PLOS ONE Jensen, J., Lysaght, A. C., Liberman, M., Qvortrup, K., Stankovic, K. M. 2015; 10 (5): e0125160

  Abstract

  Moderate acoustic overexposure in adult rodents is known to cause acute loss of synapses on sensory inner hair cells (IHCs) and delayed degeneration of the auditory nerve, despite the completely reversible temporary threshold shift (TTS) and morphologically intact hair cells. Our objective was to determine whether a cochlear synaptopathy followed by neuropathy occurs after noise exposure in pubescence, and to define neuropathic versus non-neuropathic noise levels for pubescent mice. While exposing 6 week old CBA/CaJ mice to 8-16 kHz bandpass noise for 2 hrs, we defined 97 dB sound pressure level (SPL) as the threshold for this particular type of neuropathic exposure associated with TTS, and 94 dB SPL as the highest non-neuropathic noise level associated with TTS. Exposure to 100 dB SPL caused permanent threshold shift although exposure of 16 week old mice to the same noise is reported to cause only TTS. Amplitude of wave I of the auditory brainstem response, which reflects the summed activity of the cochlear nerve, was complemented by synaptic ribbon counts in IHCs using confocal microscopy, and by stereological counts of peripheral axons and cell bodies of the cochlear nerve from 24 hours to 16 months post exposure. Mice exposed to neuropathic noise demonstrated immediate cochlear synaptopathy by 24 hours post exposure, and delayed neurodegeneration characterized by axonal retraction at 8 months, and spiral ganglion cell loss at 8-16 months post exposure. Although the damage was initially limited to the cochlear base, it progressed to also involve the cochlear apex by 8 months post exposure. Our data demonstrate a fine line between neuropathic and non-neuropathic noise levels associated with TTS in the pubescent cochlea.

  View details for DOI 10.1371/journal.pone.0125160

  View details for Web of Science ID 000356768100045

  View details for PubMedID 25955832

  View details for PubMedCentralID PMC4425526

• Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells. Cancer biology & therapy Dilwali, S., Roberts, D., Stankovic, K. M. 2015; 16 (1): 170-5

  Abstract

  Vestibular schwannoma (VS), the fourth most common intracranial tumor, arises from the Schwann cells of the vestibular nerve. Although several pathways have been independently implicated in VS pathobiology, interactions among these pathways have not been explored in depth. We have investigated the potential cross-talk between hepatocyte growth factor (HGF) and vascular endothelial growth factor-A (VEGF-A) in human VS, an interaction that has been described in other physiological and pathological cell types. We affirmed previous findings that VEGF-A signaling is aberrantly upregulated in VS, and established that expression of HGF and its receptor cMET is also significantly higher in sporadic VS than in healthy nerves. In primary human VS and Schwann cell cultures, we found that VEGF-A and HGF signaling pathways modulate each other. siRNAs targeting cMET decreased both cMET and VEGF-A protein levels, and siRNAs targeting VEGF-A reduced cMET expression. Additionally, siRNA-mediated knockdown of VEGF-A or cMET and pharmacologic inhibition of cMET decreased cellular proliferation in primary human VS cultures. Our data suggest cross-talk between these 2 prominent pathways in VS and highlight the HGF/cMET pathway as an additional important therapeutic target in VS.

  View details for DOI 10.4161/15384047.2014.972765

  View details for PubMedID 25692621

  View details for PubMedCentralID PMC4622023

• Quantitative polarized light microscopy of human cochlear sections. Biomedical optics express Low, J. C., Ober, T. J., McKinley, G. H., Stankovic, K. M. 2015; 6 (2): 599-606

  Abstract

  Dysfunction of the inner ear is the most common cause of sensorineural hearing loss, which is the most common sensory deficit worldwide. Conventional imaging modalities are unable to depict the microanatomy of the human inner ear, hence the need to explore novel imaging modalities. We provide the first characterization of the polarization dependent optical properties of human cochlear sections using quantitative polarized light microscopy (qPLM). Eight pediatric cadaveric cochlear sections, aged 0 (term) to 24 months, were selected from the US National Temporal Bone Registry, imaged with qPLM and analyzed using Image J. Retardance of the bony otic capsule and basilar membrane were substantially higher than that of the stria vascularis, spiral ganglion neurons, organ of Corti and spiral ligament across the half turns of the spiraling cochlea. qPLM provides quantitative information about the human inner ear, and awaits future exploration in vivo.

  View details for DOI 10.1364/BOE.6.000599

  View details for PubMedID 25780749

  View details for PubMedCentralID PMC4354578

• A Fully-Implantable Cochlear Implant SoC with Piezoelectric Middle-Ear Sensor and Arbitrary Waveform Neural Stimulation. IEEE journal of solid-state circuits Yip, M., Jin, R., Nakajima, H. H., Stankovic, K. M., Chandrakasan, A. P. 2015; 50 (1): 214-229

  Abstract

  A system-on-chip for an invisible, fully-implantable cochlear implant is presented. Implantable acoustic sensing is achieved by interfacing the SoC to a piezoelectric sensor that detects the sound-induced motion of the middle ear. Measurements from human cadaveric ears demonstrate that the sensor can detect sounds between 40 and 90 dB SPL over the speech bandwidth. A highly-reconfigurable digital sound processor enables system power scalability by reconfiguring the number of channels, and provides programmable features to enable a patient-specific fit. A mixed-signal arbitrary waveform neural stimulator enables energy-optimal stimulation pulses to be delivered to the auditory nerve. The energy-optimal waveform is validated with in-vivo measurements from four human subjects which show a 15% to 35% energy saving over the conventional rectangular waveform. Prototyped in a 0.18 μm high-voltage CMOS technology, the SoC in 8-channel mode consumes 572 μW of power including stimulation. The SoC integrates implantable acoustic sensing, sound processing, and neural stimulation on one chip to minimize the implant size, and proof-of-concept is demonstrated with measurements from a human cadaver ear.

  View details for DOI 10.1109/JSSC.2014.2355822

  View details for PubMedID 26251552

  View details for PubMedCentralID PMC4523309

• Functionally compromised CHD7 alleles in patients with isolated GnRH deficiency PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Balasubramanian, R., Choi, J., Francescatto, L., Willer, J., Horton, E. R., Asimacopoulos, E. P., Stankovic, K. M., Plummer, L., Buck, C. L., Quinton, R., Nebesio, T. D., Mericq, V., Merino, P. M., Meyer, B. F., Monies, D., Gusella, J. F., Al Tassan, N., Katsanis, N., Crowley, W. F. 2014; 111 (50): 17953-17958

  Abstract

  Inactivating mutations in chromodomain helicase DNA binding protein 7 (CHD7) cause CHARGE syndrome, a severe multiorgan system disorder of which Isolated gonadotropin-releasing hormone (GnRH) deficiency (IGD) is a minor feature. Recent reports have described predominantly missense CHD7 alleles in IGD patients, but it is unclear if these alleles are relevant to causality or overall genetic burden of Kallmann syndrome (KS) and normosmic form of IGD. To address this question, we sequenced CHD7 in 783 well-phenotyped IGD patients lacking full CHARGE features; we identified nonsynonymous rare sequence variants in 5.2% of the IGD cohort (73% missense and 27% splice variants). Functional analyses in zebrafish using a surrogate otolith assay of a representative set of these CHD7 alleles showed that rare sequence variants observed in controls showed no altered function. In contrast, 75% of the IGD-associated alleles were deleterious and resulted in both KS and normosmic IGD. In two families, pathogenic mutations in CHD7 coexisted with mutations in other known IGD genes. Taken together, our data suggest that rare deleterious CHD7 alleles contribute to the mutational burden of patients with both KS and normosmic forms of IGD in the absence of full CHARGE syndrome. These findings (i) implicate a unique role or preferential sensitivity for CHD7 in the ontogeny of GnRH neurons, (ii) reiterate the emerging genetic complexity of this family of IGD disorders, and (iii) demonstrate how the coordinated use of well-phenotyped cohorts, families, and functional studies can inform genetic architecture and provide insights into the developmental biology of cellular systems.

  View details for DOI 10.1073/pnas.1417438111

  View details for Web of Science ID 000346366500073

  View details for PubMedID 25472840

  View details for PubMedCentralID PMC4273325

• A 1.1nW Energy Harvesting System with 544pW Quiescent Power for Next Generation Implants. IEEE journal of solid-state circuits Bandyopadhyay, S., Mercier, P. P., Lysaght, A. C., Stankovic, K. M., Chandrakasan, A. P. 2014; 49 (12): 2812-2824

  Abstract

  This paper presents a nW power management unit (PMU) for an autonomous wireless sensor that sustains itself by harvesting energy from the endocochlear potential (EP), the 70-100 mV electrochemical bio-potential inside the mammalian ear. Due to the anatomical constraints inside the inner ear, the total extractable power from the EP is limited to 1.1-6.25 nW. A nW boost converter is used to increase the input voltage (30-55 mV) to a higher voltage (0.8 to 1.1 V) usable by CMOS circuits in the sensor. A pW Charge Pump circuit is used to minimize the leakage in the boost converter. Further, ultra-low-power control circuits consisting of digital implementations of input impedance adjustment circuits and Zero Current Switching circuits along with Timer and Reference circuits keep the quiescent power of the PMU down to 544 pW. The designed boost converter achieves a peak power conversion efficiency of 56%. The PMU can sustain itself and a duty-cyled ultra-low power load while extracting power from the EP of a live guinea pig. The PMU circuits have been implemented on a 0.18µm CMOS process.

  View details for DOI 10.1109/JSSC.2014.2350260

  View details for PubMedID 25983340

  View details for PubMedCentralID PMC4428497

• Cochlin in normal middle ear and abnormal middle ear deposits in DFNA9 and Coch (G88E/G88E) mice. Journal of the Association for Research in Otolaryngology : JARO Robertson, N. G., O'Malley, J. T., Ong, C. A., Giersch, A. B., Shen, J., Stankovic, K. M., Morton, C. C. 2014; 15 (6): 961-74

  Abstract

  DFNA9 sensorineural hearing loss and vestibular disorder, caused by mutations in COCH, has a unique identifying histopathology including prominent acellular deposits in cochlear and vestibular labyrinths. A recent study has shown presence of deposits also in middle ear structures of DFNA9-affected individuals (McCall et al., J Assoc Res Otolaryngol 12:141-149, 2004). To investigate the possible role of cochlin in the middle ear and in relation to aggregate formation, we evaluated middle ear histopathology in our Coch knock-in (Coch (G88E/G88E) ) mouse model, which harbors one of the DFNA9-causative mutations. Our findings reveal accumulation of acellular deposits in the incudomalleal and incudostapedial joints in Coch (G88E/G88E) mice, similar to those found in human DFNA9-affected temporal bones. Aggregates are absent in negative control Coch (+/+) and Coch (-/-) mice. Thickening of the tympanic membrane (TM) found in humans with DFNA9 was not appreciably detected in Coch (G88E/G88E) mice at the evaluated age. We investigated cochlin localization first in the Coch (+/+)mouse and in normal human middle ears, and found prominent and specific cochlin staining in the incudomalleal joint, incudostapedial joint, and the pars tensa of the TM, which are the three sites where abnormal deposits are detected in DFNA9-affected middle ears. Cochlin immunostaining of Coch (G88E/G88E) and DFNA9-affected middle ears showed mutant cochlin localization within areas of aggregates. Cochlin staining was heterogeneous throughout DFNA9 middle ear deposits, which appear as unorganized and overlapping mixtures of both eosinophilic and basophilic substances. Immunostaining for type II collagen colocalized with cochlin in pars tensa of the tympanic membrane. In contrast, immunostaining for type II collagen did not overlap with cochlin in interossicular joints, where type II collagen was localized in the region of the chondrocytes, but not in the thin layer of the articular surface of the ossicles nor in the eosinophilic deposits with specific cochlin staining.

  View details for DOI 10.1007/s10162-014-0481-9

  View details for PubMedID 25049087

  View details for PubMedCentralID PMC4389958

• Primary culture of human Schwann and schwannoma cells: Improved and simplified, protocol HEARING RESEARCH Dilwali, S., Patel, P. B., Roberts, D. S., Basinsky, G. M., Harris, G. J., Emerick, K. S., Stankovic, K. M. 2014; 315: 25-33

  Abstract

  Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology.

  View details for DOI 10.1016/j.heares.2014.05.006

  View details for Web of Science ID 000341902700003

  View details for PubMedID 24910344

  View details for PubMedCentralID PMC4164296

• A Sub-nW 2.4 GHz Transmitter for Low Data-Rate Sensing Applications IEEE JOURNAL OF SOLID-STATE CIRCUITS Mercier, P. P., Bandyopadhyay, S., Lysaght, A. C., Stankovic, K. M., Chandrakasan, A. P. 2014; 49 (7): 1463-1474

  Abstract

  This paper presents the design of a narrowband transmitter and antenna system that achieves an average power consumption of 78 pW when operating at a duty-cycled data rate of 1 bps. Fabricated in a 0.18 µm CMOS process, the transmitter employs a direct-RF power oscillator topology where a loop antenna acts as a both a radiative and resonant element. The low-complexity single-stage architecture, in combination with aggressive power gating techniques and sizing optimizations, limited the standby power of the transmitter to only 39.7 pW at 0.8 V. Supporting both OOK and FSK modulations at 2.4 GHz, the transmitter consumed as low as 38 pJ/bit at an active-mode data rate of 5 Mbps. The loop antenna and integrated diodes were also used as part of a wireless power transfer receiver in order to kick-start the system power supply during energy harvesting operation.

  View details for DOI 10.1109/JSSC.2014.2316237

  View details for Web of Science ID 000338512700002

  View details for PubMedID 26246641

  View details for PubMedCentralID PMC4523311

• Mining Immune Epitopes in the Inner Ear OTOLARYNGOLOGY-HEAD AND NECK SURGERY Platt, M., Dilwali, S., Elackattu, A., Parikh, J. R., Stankovic, K. M. 2014; 150 (3): 460-463

  Abstract

  Etiologies for many inner ear disorders, including autoimmune inner ear disease, sudden sensorineural hearing loss, and Meniere's disease, remain unknown. Indirect evidence suggests an immune-mediated process involving an allergic or autoimmune mechanism. We examined whether known immunogenic proteins share sequence similarity with inner ear proteins, which may lead to cross-reactivity and detrimental immune activation. Comprehensive bioinformatic analyses of primary sequences of intact and mutated proteins associated with human hearing loss and all proteins known to be expressed in the human inner ear were compared with all immune epitopes in the Immune Epitope Database. The exact match and basic local alignment search tool computational algorithms identified 3036 and 106 unique epitope matches, respectively, the majority of which were infectious epitopes. If validated in future clinical trials, these candidate immune epitopes in the inner ear would be potential novel targets for diagnosis and treatment of some inner ear disorders and the resulting hearing loss.

  View details for DOI 10.1177/0194599813514725

  View details for Web of Science ID 000331705800022

  View details for PubMedID 24367049

• Aspirin Intake Correlates With Halted Growth of Sporadic Vestibular Schwannoma In Vivo OTOLOGY & NEUROTOLOGY Kandathil, C. K., Dilwali, S., Wu, C., Ibrahimov, M., McKenna, M. J., Lee, H., Stankovic, K. M. 2014; 35 (2): 353-357

  Abstract

  Given the presence of a pathological immune response in sporadic vestibular schwannoma (sVS), this study aims to explore the roles of aspirin in minimizing sVS growth in vivo.Retrospective case review.Tertiary care hospital.People diagnosed with sVS and followed at a tertiary referral center by serial magnetic resonance imaging (MRI) for at least 4 months within the period of January 1980 through April 2012.Patient use of aspirin and sVS growth rate measured by changes in the largest tumor dimension as noted on serial MRIsWithin a set of 689 cases, 347 were followed by serial MRI scans (50.3%); of the latter, 81 took aspirin, of which, 33 demonstrated sVS growth, and 48 did not. Of the 266 nonaspirin users, 154 demonstrated sVS growth, and 112 did not. A significant inverse association was found among aspirin users and sVS growth (odds ratio [OR]: 0.50, 95% confidence interval [CI]: 0.29-0.85), which was not confounded by age or sex.Our results suggest a potential therapeutic role of aspirin in inhibiting sVS growth.

  View details for DOI 10.1097/MAO.0000000000000189

  View details for Web of Science ID 000337697100033

  View details for PubMedID 24448296

• FGF23 deficiency leads to mixed hearing loss and middle ear malformation in mice. PloS one Lysaght, A. C., Yuan, Q. n., Fan, Y. n., Kalwani, N. n., Caruso, P. n., Cunnane, M. n., Lanske, B. n., Stanković, K. M. 2014; 9 (9): e107681

  Abstract

  Fibroblast growth factor 23 (FGF23) is a circulating hormone important in phosphate homeostasis. Abnormal serum levels of FGF23 result in systemic pathologies in humans and mice, including renal phosphate wasting diseases and hyperphosphatemia. We sought to uncover the role FGF23 plays in the auditory system due to shared molecular mechanisms and genetic pathways between ear and kidney development, the critical roles multiple FGFs play in auditory development and the known hearing phenotype in mice deficient in klotho (KL), a critical co-factor for FGF23 signaling. Using functional assessments of hearing, we demonstrate that Fgf[Formula: see text] mice are profoundly deaf. Fgf[Formula: see text] mice have moderate hearing loss above 20 kHz, consistent with mixed conductive and sensorineural pathology of both middle and inner ear origin. Histology and high-voltage X-ray computed tomography of Fgf[Formula: see text] mice demonstrate dysplastic bulla and ossicles; Fgf[Formula: see text] mice have near-normal morphology. The cochleae of mutant mice appear nearly normal on gross and microscopic inspection. In wild type mice, FGF23 is ubiquitously expressed throughout the cochlea. Measurements from Fgf[Formula: see text] mice do not match the auditory phenotype of Kl-/- mice, suggesting that loss of FGF23 activity impacts the auditory system via mechanisms at least partially independent of KL. Given the extensive middle ear malformations and the overlap of initiation of FGF23 activity and Eustachian tube development, this work suggests a possible role for FGF23 in otitis media.

  View details for DOI 10.1371/journal.pone.0107681

  View details for PubMedID 25243481

  View details for PubMedCentralID PMC4171482

• A 1.1nW Energy Harvesting System with 544pW Quiescent Power for Next-Generation Implants Bandyopadhyay, S., Mercier, P. P., Lysaght, A. C., Stankovic, K. M., Chandrakasan, A. P., IEEE IEEE. 2014: 396-+

  View details for Web of Science ID 000353615000163

• Metabolomic and Network Analysis of Pharmacotherapies for Sensorineural Hearing Loss OTOLOGY & NEUROTOLOGY Muurling, T., Stankovic, K. M. 2014; 35 (1): 1-6

  Abstract

  Different pharmacotherapies for sensorineural hearing loss (SNHL) are interconnected in metabolic networks with molecular hubs.Sensorineural hearing loss is the most common sensory deficit worldwide. Dozens of drugs have shown efficacy against SNHL in animal studies and a few in human studies. Analyzing metabolic networks that interconnect these drugs will point to and prioritize development of new pharmacotherapies for human SNHL.Drugs that have shown efficacy in treating mammalian SNHL were identified through PubMed literature searches. The drugs were analyzed using the metabolomic analysis and the "grow-tool function" in ingenuity pathway analysis (IPA). The top 3 most interconnected molecules and drugs (i.e., the hubs) within the generated networks were considered important targets for the treatment of SNHL.A total of 70 drugs were investigated with IPA. The metabolomic analysis revealed 2 statistically significant networks (Networks 1 and 2). A network analysis using the "grow-tool function" generated one statistically significant network (Network 3). Hubs of these networks were as follows: P38 mitogen-activated protein kinases (P38 MAPK), p42/p44 MAP kinase (ERK1/2) and glutathione for Network 1; protein kinase B (Akt), nuclear factor kappa B (NFkB) and ERK for Network 2; and dexamethasone, tretinoin, and cyclosporin A for Network 3.Metabolomic and network analysis of the existing pharmacotherapies for SNHL has pointed to and prioritized a number of potential novel targets for treatment of SNHL.

  View details for Web of Science ID 000337694300013

  View details for PubMedID 24335929

• Loss of osteoprotegerin expression in the inner ear causes degeneration of the cochlear nerve and sensorineural hearing loss NEUROBIOLOGY OF DISEASE Kao, S., Kempfle, J. S., Jensen, J. B., Perez-Fernandez, D., Lysaght, A. C., Edge, A. S., Stankovic, K. M. 2013; 56: 25-33

  Abstract

  Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations and variations in the OPG gene cause many human diseases that are characterized by not only skeletal abnormalities but also poorly understood hearing loss: Paget's disease, osteoporosis, and celiac disease. To gain insight into the mechanisms of hearing loss in OPG deficiency, we studied OPG knockout (Opg(-/-)) mice. We show that they develop sensorineural hearing loss, in addition to conductive hearing loss due to abnormal middle-ear bones. OPG deficiency caused demyelination and degeneration of the cochlear nerve in vivo. It also activated ERK, sensitized spiral ganglion cells (SGC) to apoptosis, and inhibited proliferation and survival of cochlear stem cells in vitro, which could be rescued by treatment with exogenous OPG, an ERK inhibitor, or bisphosphonate. Our results demonstrate a novel role for OPG in the regulation of SGC survival, and suggest a mechanism for sensorineural hearing loss in OPG deficiency.

  View details for DOI 10.1016/j.nbd.2013.04.008

  View details for Web of Science ID 000323853300004

  View details for PubMedID 23607938

  View details for PubMedCentralID PMC3752395

• A Comprehensive Network and Pathway Analysis of Human Deafness Genes OTOLOGY & NEUROTOLOGY Stamatiou, G. A., Stankovic, K. M. 2013; 34 (5): 961-970

  Abstract

  To perform comprehensive network and pathway analyses of the genes known to cause genetic hearing loss.In silico analysis of deafness genes using ingenuity pathway analysis (IPA).Genes relevant for hearing and deafness were identified through PubMed literature searches and the Hereditary Hearing Loss Homepage. The genes were assembled into 3 groups: 63 genes that cause nonsyndromic deafness, 107 genes that cause nonsyndromic or syndromic sensorineural deafness, and 112 genes associated with otic capsule development and malformations. Each group of genes was analyzed using IPA to discover the most interconnected, that is, "nodal" molecules, within the most statistically significant networks (p < 10).The number of networks that met our criterion for significance was 1 for Group 1 and 2 for Groups 2 and 3. Nodal molecules of these networks were as follows: transforming growth factor beta1 (TGFB1) for Group 1, MAPK3/MAPK1 MAP kinase (ERK 1/2) and the G protein coupled receptors (GPCR) for Group 2, and TGFB1 and hepatocyte nuclear factor 4 alpha (HNF4A) for Group 3. The nodal molecules included not only those known to be associated with deafness (GPCR), or with predisposition to otosclerosis (TGFB1), but also novel genes that have not been described in the cochlea (HNF4A) and signaling kinases (ERK 1/2).A number of molecules that are likely to be key mediators of genetic hearing loss were identified through three different network and pathway analyses. The molecules included new candidate genes for deafness. Therapies targeting these molecules may be useful to treat deafness.

  View details for Web of Science ID 000330371400032

  View details for PubMedID 23770690

• Sporadic Vestibular Schwannomas Associated With Good Hearing Secrete Higher Levels of Fibroblast Growth Factor 2 Than Those Associated With Poor Hearing Irrespective of Tumor Size OTOLOGY & NEUROTOLOGY Dilwali, S., Lysaght, A., Roberts, D., Barker, F. G., McKenna, M. J., Stankovic, K. M. 2013; 34 (4): 748-754

  Abstract

  We hypothesize that the severity of hearing loss (HL) associated with sporadic vestibular schwannomas (VS) is correlated with tumor secretion of proteins with ototoxic or otoprotective potential.Because the recognition that HL associated with VS is not solely due to compression of the auditory nerve, elucidating the mechanism by which VS cause HL has been an important task. We previously showed that VS stratified by hearing have differential gene expression. We now focus on identifying differentially expressed proteins in tumor secretions.Fresh surgical specimens of VS were incubated in sterile PBS at 37°C to collect secretions. The specimens were divided into a group associated with good hearing (GH, word recognition ≥ 70% and pure-tone average ≤ 30 dB, n = 11) or poor hearing (PH, n = 10). The groups were compared using a customized cytokine array. Statistically significant results were verified with an enzyme-linked immunosorbent assay on a different set of secretions (n = 8 for GH and n = 10 for PH group).Of the 37 molecules we studied, 9 were significantly expressed in secretions from VS compared with secretions from control nerves. Secretion of fibroblast growth factor 2 (FGF2) was 3.5-fold higher in VS associated with GH versus PH based on cytokine array analysis (p = 0.02), which was validated with enzyme-linked immunosorbent assay.This study highlights FGF2, a mitogen known to protect the auditory nerve, as a potential tumor-secreted mediator of hearing protection in VS. If FGF2's significant role in hearing protection in patients with VS is validated, then FGF2 could be used as a biomarker for HL in VS, and therapeutic targeting of the FGF2 signaling pathway may reduce HL due to VS.

  View details for DOI 10.1097/MAO.0b013e31828048ec

  View details for Web of Science ID 000319062100030

  View details for PubMedID 23512073

  View details for PubMedCentralID PMC3655133

• Two-photon microscopy of the mouse cochlea in situ for cellular diagnosis JOURNAL OF BIOMEDICAL OPTICS Yang, X., Pu, Y., Hsieh, C., Ong, C., Psaltis, D., Stankovic, K. M. 2013; 18 (3): 31104

  Abstract

  Sensorineural hearing loss is the most common type of hearing loss worldwide, yet the underlying cause is typically unknown because the inner ear cannot be biopsied today without destroying hearing, and intracochlear cells have not been imaged with resolution sufficient to establish diagnosis. Intracochlear imaging has been technologically challenging because of the cochlea's small size and encasement in bone. We report, for the first time, imaging of the mouse cochlea in situ without exogenous dyes, through a membranous round window, using a near-infrared femtosecond laser as the excitation and endogenous two-photon excitation fluorescence (TPEF) and second harmonic generation as the contrast mechanisms. We find that TPEF exhibits strong contrast allowing cellular, and even subcellular resolution, and detection of specific, noise-induced pathologic changes. Our results demonstrate that the round window provides a useful access to the cochlea through the middle ear, and they motivate future development of a new and efficient diagnostic tool based on two-photon micro-endoscopy.

  View details for DOI 10.1117/1.JBO.18.3.031104

  View details for Web of Science ID 000322336700008

  View details for PubMedID 23165736

• Case records of the Massachusetts General Hospital. Case 2-2013. A 20-year-old man with recurrent ear pain, fever, and headache. The New England journal of medicine Stankovic, K. M., Eskandar, E., El Khoury, J. B., Lev, M. H., Sadow, P. M. 2013; 368 (3): 267-77

  View details for DOI 10.1056/NEJMcpc1200089

  View details for PubMedID 23323904

• Quantitative polarized light microscopy of unstained mammalian cochlear sections. Journal of biomedical optics Kalwani, N. M., Ong, C. A., Lysaght, A. C., Haward, S. J., McKinley, G. H., Stankovic, K. M. 2013; 18 (2): 26021

  Abstract

  Hearing loss is the most common sensory deficit in the world, and most frequently it originates in the inner ear. Yet, the inner ear has been difficult to access for diagnosis because of its small size, delicate nature, complex three-dimensional anatomy, and encasement in the densest bone in the body. Evolving optical methods are promising to afford cellular diagnosis of pathologic changes in the inner ear. To appropriately interpret results from these emerging technologies, it is important to characterize optical properties of cochlear tissues. Here, we focus on that characterization using quantitative polarized light microscopy (qPLM) applied to unstained cochlear sections of the mouse, a common animal model of human hearing loss. We find that the most birefringent cochlear materials are collagen fibrils and myelin. Retardance of the otic capsule, the spiral ligament, and the basilar membrane are substantially higher than that of other cochlear structures. Retardance of the spiral ligament and the basilar membrane decrease from the cochlear base to the apex, compared with the more uniform retardance of other structures. The intricate structural details revealed by qPLM of unstained cochlear sections ex vivo strongly motivate future application of polarization-sensitive optical coherence tomography to human cochlea in vivo.

  View details for DOI 10.1117/1.JBO.18.2.026021

  View details for PubMedID 23407909

  View details for PubMedCentralID PMC3571355

• Energy extraction from the biologic battery in the inner ear NATURE BIOTECHNOLOGY Mercier, P. P., Lysaght, A. C., Bandyopadhyay, S., Chandrakasan, A. P., Stankovic, K. M. 2012; 30 (12): 1240-+

  Abstract

  Endocochlear potential (EP) is a battery-like electrochemical gradient found in and actively maintained by the inner ear. Here we demonstrate that the mammalian EP can be used as a power source for electronic devices. We achieved this by designing an anatomically sized, ultra-low quiescent-power energy harvester chip integrated with a wireless sensor capable of monitoring the EP itself. Although other forms of in vivo energy harvesting have been described in lower organisms, and thermoelectric, piezoelectric and biofuel devices are promising for mammalian applications, there have been few, if any, in vivo demonstrations in the vicinity of the ear, eye and brain. In this work, the chip extracted a minimum of 1.12 nW from the EP of a guinea pig for up to 5 h, enabling a 2.4 GHz radio to transmit measurement of the EP every 40-360 s. With future optimization of electrode design, we envision using the biologic battery in the inner ear to power chemical and molecular sensors, or drug-delivery actuators for diagnosis and therapy of hearing loss and other disorders.

  View details for DOI 10.1038/nbt.2394

  View details for Web of Science ID 000312092400028

  View details for PubMedID 23138225

  View details for PubMedCentralID PMC3938019

• Sensorineural Hearing Loss following Imatinib (Gleevec) Administration OTOLARYNGOLOGY-HEAD AND NECK SURGERY Lin, H. W., Roberts, D. S., Kay, J., Stankovic, K. M. 2012; 146 (2): 335-337

  View details for DOI 10.1177/0194599811415008

  View details for Web of Science ID 000303541300031

  View details for PubMedID 21753034

• Proteome of Human Perilymph JOURNAL OF PROTEOME RESEARCH Lysaght, A. C., Kao, S., Paulo, J. A., Merchant, S. N., Steen, H., Stankovic, K. M. 2011; 10 (9): 3845-3851

  Abstract

  Current diagnostic tools limit a clinician's ability to discriminate between many possible causes of sensorineural hearing loss. This constraint leads to the frequent diagnosis of the idiopathic condition, leaving patients without a clear prognosis and only general treatment options. As a first step toward developing new diagnostic tools and improving patient care, we report the first use of liquid chromatography-tandem mass-spectrometry (LC-MS/MS) to map the proteome of human perilymph. Using LC-MS/MS, we analyzed four samples, two collected from patients with vestibular schwannoma (VS) and two from patients undergoing cochlear implantation (CI). For each cohort, one sample contained pooled specimens collected from five patients and the second contained a specimen obtained from a single patient. Of the 271 proteins identified with high confidence among the samples, 71 proteins were common in every sample and used to conservatively define the proteome of human perilymph. Comparison to human cerebrospinal fluid and blood plasma, as well as murine perilymph, showed significant similarity in protein content across fluids; however, a quantitative comparison was not possible. Fifteen candidate biomarkers of VS were identified by comparing VS and CI samples. This list will be used in future investigations targeted at discriminating between VS tumors associated with good versus poor hearing.

  View details for DOI 10.1021/pr200346q

  View details for Web of Science ID 000294446600001

  View details for PubMedID 21740021

  View details for PubMedCentralID PMC3179892

• Topographic Gene Expression in the Sinonasal Cavity of Patients with Chronic Sinusitis with Polyps OTOLARYNGOLOGY-HEAD AND NECK SURGERY Platt, M. P., Soler, Z. M., Kao, S., Metson, R., Stankovic, K. M. 2011; 145 (1): 171-175

  Abstract

  To determine whether variations in gene expression exist at multiple subsites along the sinonasal tract in patients with chronic sinusitis with polyps and in healthy controls.Prospective, controlled study.Academic medical center.Tissue expression levels of 5 genes, previously found to be characteristic of ethmoid polyps, were measured using real-time quantitative polymerase chain reaction in 100 sinonasal tissue samples. Specimens harvested from 5 regions--the ethmoid sinus, septum, inferior turbinate, middle turbinate, and lateral nasal wall--in 10 patients with chronic sinusitis and ethmoid polyps were compared to tissue from similar regions in 10 control patients without sinusitis. Western blot analysis was performed to validate differential gene expression at the protein level.Gene expression levels of ethmoid polyps differed significantly from those of healthy ethmoid mucosa, as well as tissue from 4 surrounding anatomical sites in both patients with chronic sinusitis and controls. Alterations specific to the polyp tissue included downregulated genes, prolactin-induced protein (fold change 377.2 ± 169.0, P < .0001), and zinc α2-glycoprotein (fold change 72.1 ± 26.5, P < .0001), as well as upregulated genes, met proto-oncogene (fold change 2.5 ± 0.7, P = .029), and periostin (fold change 7.5 ± 3.4, P = .003). No significant differences in gene expression was found for neurabin 2 (fold change 1.0, P = .99).The transcriptional pattern of ethmoid polyps appears to be unique compared with other subsites in the sinonasal cavity of patients with chronic sinusitis. Care must be taken when collecting specimens for molecular studies of the sinonasal tract to differentiate polyp from nonpolyp tissue in chronic sinusitis.

  View details for DOI 10.1177/0194599811402030

  View details for Web of Science ID 000293999000027

  View details for PubMedID 21493287

  View details for PubMedCentralID PMC3752390

• Pathways analysis of molecular markers in chronic sinusitis with polyps. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery Platt, M. P., Soler, Z., Metson, R., Stankovic, K. M. 2011; 144 (5): 802-8

  Abstract

  To perform a comprehensive molecular pathways analysis of genes identified through genome-wide expression profiling and the published literature for chronic sinusitis with polyps.Molecular pathways analysis.Academic medical center.A molecular pathways analysis of gene biomarkers discovered through hypothesis-driven and high-throughput molecular studies was performed. Genes identified with a PubMed literature search were analyzed with Ingenuity Pathways Analysis software to identify central molecules implicated in the pathogenesis of chronic sinusitis with polyps. The central pathways were then compared with those identified through genome-wide expression profiling of ethmoid polyps.A total of 97 molecules were investigated with Ingenuity Pathways Analysis based on 55 studies that evaluated differences in gene expression (39), genetic variation (12), or proteomics (4). The analysis revealed 9 statistically significant molecular networks containing central nodes that included transcription factors, protein kinases, cytokines, and growth factors/receptors. The highest scoring networks implicated nuclear factor kappa-B, tumor necrosis factor, and mitogen-activated protein kinases. The majority of pathways in the literature review analysis overlapped with those identified through a single genome-wide expression study.Chronic sinusitis with polyps is a complex disease with suspected contribution of multiple genetic and environmental factors. The search for causative genes has led to the discovery of numerous candidates. Pathways analysis applied to these candidate genes identified common central molecules that are likely to be key mediators of the disease process. Novel therapies targeting these molecules may be applicable for the treatment of chronic sinusitis with polyps.

  View details for DOI 10.1177/0194599810395091

  View details for PubMedID 21493366

  View details for PubMedCentralID PMC4164309

• Cochlear implantation in children with congenital X-linked deafness due to novel mutations in POU3F4 gene. The Annals of otology, rhinology, and laryngology Stankovic, K. M., Hennessey, A. M., Herrmann, B., Mankarious, L. A. 2010; 119 (12): 815-22

  Abstract

  We report novel mutations in the POU3F4 gene resulting in congenital X-linked deafness DFN3, and describe the results of cochlear implantation in 4 boys (3 siblings) followed for an average of 3.5 years.The diagnosis of DFN3 was made in infant boys on the basis of the radiologic criteria of an underdeveloped modiolus, a wide cochlear fossette, and the presence of all cochlear turns. The POU3F4 gene was sequenced. A standard, transmastoid, facial recess approach was used for cochlear implantation. A lumbar drain was placed before the operation.The identified mutations in the POU3F4 gene were novel (p.R167X in the 3 siblings) or recently reported (p.S310del). A high-flow cerebrospinal fluid leak through the cochleostomy was encountered in each patient and was ultimately controlled. Although the implants functioned properly, the auditory perceptual abilities did not progress past sound detection in the 3 siblings, or past closed-set word identification in the non-sibling, who achieved better speech perception with contralateral amplification. Three boys (2 siblings) show signs of other learning disorders; 1 boy was too young for a complete assessment.Preoperative gene mutation analysis in DFN3 patients who are considering cochlear implantation may help in long-term counseling and in avoidance of postoperative complications. Limited auditory perception and language acquisition may result. Amplification may sometimes be a better alternative than cochlear implantation, despite the severity of the hearing loss.

  View details for DOI 10.1177/000348941011901205

  View details for PubMedID 21250553

• Case records of the Massachusetts General Hospital. Case 36-2010. A 50-year-old woman with pain and loss of hearing in the left ear. The New England journal of medicine Stankovic, K. M., Juliano, A. F., Hasserjian, R. P. 2010; 363 (22): 2146-56

  View details for DOI 10.1056/NEJMcpc1000967

  View details for PubMedID 21105797

• Nonneuronal cells regulate synapse formation in the vestibular sensory epithelium via erbB-dependent BDNF expression PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gomez-Casati, M. E., Murtie, J. C., Rio, C., Stankovic, K., Liberman, M., Corfas, G. 2010; 107 (39): 17005-17010

  Abstract

  Recent studies indicate that molecules released by glia can induce synapse formation. However, what induces glia to produce such signals, their identity, and their in vivo relevance remain poorly understood. Here we demonstrate that supporting cells of the vestibular organ--cells that have many characteristics of glia--promote synapse formation only when induced by neuron-derived signals. Furthermore, we identify BDNF as the synaptogenic signal produced by these nonneuronal cells. Mice in which erbB signaling has been eliminated in supporting cells have vestibular dysfunction caused by failure of synapse formation between hair cells and sensory neurons. This phenotype correlates with reduced BDNF expression in supporting cells and is rescued by reexpression of BDNF in these cells. Furthermore, knockdown of BDNF expression in supporting cells postnatally phenocopies the loss of erbB signaling. These results indicate that vestibular supporting cells contribute in vivo to vestibular synapse formation and that this is mediated by reciprocal signals between sensory neurons and supporting cells involving erbB receptors and BDNF.

  View details for DOI 10.1073/pnas.1008938107

  View details for Web of Science ID 000282211700050

  View details for PubMedID 20837532

  View details for PubMedCentralID PMC2947909

• Differences in gene expression between the otic capsule and other bones HEARING RESEARCH Stankovic, K. M., Adachi, O., Tsuji, K., Kristiansen, A. G., Adams, J. C., Rosen, V., McKenna, M. J. 2010; 265 (1-2): 83-89

  Abstract

  Our long term goal is to understand the molecular pathology of otosclerosis and to develop better forms of therapy. Toward this goal, the current study focused on characterizing the molecular factors responsible for the unique biological features of the otic capsule: its minimal rate of remodeling, and lack of healing capacity when fractured. We compared expression levels of 62 genes involved in bone metabolism between the adult murine otic capsule and the tibia and parietal bones; the latter exemplify bones formed by endochondral and intramembranous ossification, respectively. Gene expression levels were measured using real-time quantitative RT-PCR and analyzed using tools of bioinformatics. Expression patterns of key genes were verified with in situ hybridization. The molecular profile of the otic capsule was distinctly different from that of the tibia and parietal bone. Genes found to be most characteristic of the otic capsule were: osteoprotegerin (opg), bone morphogenetic protein receptor 1b (bmpr1b) and bone morphogenetic protein 3 (bmp3). Expression levels were high for opg and bmpr1b, and minimal for bmp3 within the otic capsule. We concluded that opg and bmpr1b likely play important roles in inhibition of remodeling within the otic capsule.

  View details for DOI 10.1016/j.heares.2010.02.006

  View details for Web of Science ID 000279092000012

  View details for PubMedID 20146935

  View details for PubMedCentralID PMC3586807

• Genetic Determinants of Hearing Loss Associated With Vestibular Schwannomas OTOLOGY & NEUROTOLOGY Stankovic, K. M., Mrugala, M. M., Martuza, R. L., Silver, K., Betensky, K. A., Nadol, J. B., Stemmer-Rachamimov, A. O. 2009; 30 (5): 661-667

  Abstract

  The severity of hearing loss (HL) associated with vestibular schwannomas (VSs) is influenced by genes expressed by the VSs.Hearing loss is the most common presenting symptoms in patients with VSs, yet its pathophysiology remains elusive. Previous studies have suggested that VSs cause HL not only by inducing degeneration of the auditory nerve by compression but also by promoting degeneration of the inner ear. This study aimed to determine whether there is a molecular basis for differences in HL associated with VSs.Surgical specimens of VSs were collected from 13 patients and were divided into a group associated with good (word recognition >70% and pure-tone average < or =30 dB) or poor hearing. Whole-genome expression profiling of VSs was performed with the Affymetrix GeneChip Human X3P Array. The expression of select genes was validated using real-time quantitative reverse transcription-polymerase chain reaction and immunohistochemistry. Because of a small sample size, exact nonparametric tests were used to assess the association between good versus poor hearing and specific histological features of the tumors and patient demographics.Using gene set enrichment analysis, the chromosomal region 3q27 was found to be significantly different between the 2 groups of tumors. This region includes peroxisomal biogenesis factor 5-like gene, which was underexpressed in VSs with poor hearing. The expression of 3 other genes from different chromosomes was significantly different between the 2 groups: RAD54B, prostate-specific membrane antigen-like, and carcinoembryonic antigen.This study identified several molecular alterations in VSs stratified by hearing. These alterations may determine the severity of HL associated with VSs and may represent potential therapeutic targets to prevent or reduce HL in theses patients.

  View details for DOI 10.1097/MAO.0b013e3181a66ece

  View details for Web of Science ID 000276926400016

  View details for PubMedID 19546833

• Gene-expression signatures of nasal polyps associated with chronic rhinosinusitis and aspirin-sensitive asthma. Current opinion in allergy and clinical immunology Platt, M., Metson, R., Stankovic, K. 2009; 9 (1): 23-8

  Abstract

  The purpose of this review is to highlight recent advances in gene-expression profiling of nasal polyps in patients with chronic rhinosinusitis and aspirin-sensitive asthma.Gene-expression profiling has allowed simultaneous interrogation of thousands of genes, including the entire genome, to better understand distinct biological and clinical phenotypes associated with nasal polyps. The genes with altered expression in nasal polyps are involved in many cellular processes, including growth and development, immune functions, and signal transduction. The wide-ranging and typically nonoverlapping results reported in the published studies reflect methodological and demographic differences. The identified genes present possible novel therapeutic targets for nasal polyps associated with chronic rhinosinusitis and aspirin-sensitive asthma.Gene-expression profiling is a powerful technology that allows definition of expression signatures to characterize patient subgroups, predict response to treatment, and offer novel therapies. Although the ability to interpret the meaning of the individual gene in these signatures remains a challenge, integrated analysis of a large number of these signatures with other genome-scale data sets and more traditional targeted approaches has a potential to revolutionarize understanding and treatment of chronic rhinosinusitis and aspirin-sensitive asthma.

  View details for DOI 10.1097/ACI.0b013e32831d8170

  View details for PubMedID 19532090

• Gene Expression Profiling of Nasal Polyps Associated With Chronic Sinusitis and Aspirin-Sensitive Asthma LARYNGOSCOPE Stankovic, K. M., Goldsztein, H., Reh, D. D., Platt, M. P., Metson, R. 2008; 118 (5): 881-889

  Abstract

  To identify genes whose expression is most characteristic of chronic rhinosinusitis and aspirin-sensitive asthma through genome-wide transcriptional profiling of nasal polyp tissue.Prospective, controlled study conducted at a tertiary care institution.Thirty genome-wide expression microarrays were used to compare nasal polyp tissue from patients with chronic rhinosinusitis alone (CRS, n = 10) or chronic rhinosinusitis and a history of aspirin-sensitive asthma (ASA, n = 10) to normal sinonasal mucosa from patients who underwent surgery for non-sinus related conditions (controls, n = 10). Genes found to be most characteristic of each polyp phenotype, as determined from bioinformatic analyses, were validated using real-time quantitative polymerase chain reaction (RT-PCR) and immunohistochemistry in different patient sets.The transcriptional signature of the control mucosa was distinctly different from that of either polyp phenotype. Genes most characteristic of the CRS phenotype included two upregulated genes--met proto-oncogene (MET) and protein phosphatase 1 regulatory subunit 9B (PPP1R9B)-and two downregulated genes--prolactin-induced protein (PIP) and zinc alpha2-glycoprotein (AZGP1). The gene most characteristic of the ASA phenotype was periostin (POSTN), which was upregulated relative to controls. Differences between the CRS and ASA phenotypes were associated with alterations in the 6p22, 22q13, and 1q23 chromosomal regions.Nasal polyps appear to have characteristic transcriptional signatures compared to normal sinonasal mucosa. The five genes identified in this study likely play roles in the pathogenesis of polyps associated with CRS and ASA, and are therefore attractive targets for novel medical therapies for these common debilitating diseases.

  View details for DOI 10.1097/MLG.0b013e31816b4b6f

  View details for Web of Science ID 000260662100022

  View details for PubMedID 18391768

• Dynamic patterns of neurotrophin 3 expression in the postnatal mouse inner ear. The Journal of comparative neurology Sugawara, M., Murtie, J. C., Stankovic, K. M., Liberman, M. C., Corfas, G. 2007; 501 (1): 30-7

  Abstract

  Recent studies indicate that neurotrophin 3 (NT3) may be important for the maintenance and function of the adult inner ear, but the pattern of postnatal NT3 expression in this organ has not been characterized. We used a reporter mouse in which cells expressing NT3 also express beta-galactosidase, allowing for their histochemical visualization, to determine the pattern of NT3 expression in cochlear and vestibular organs. We analyzed animals from birth (P0) to adult (P135). At P0, NT3 was strongly expressed in supporting cells and hair cells of all vestibular and cochlear sense organs, Reissner's membrane, saccular membrane, and the dark cells adjacent to canal organs. With increasing age, staining disappeared in most cell types but remained relatively high in inner hair cells (IHCs) and to a lesser extent in IHC supporting cells. In the cochlea, by P0 there is a longitudinal gradient (apex > base) that persists into adulthood. In vestibular maculae, staining gradients are: striolar > extrastriolar regions and supporting cells > hair cells. By P135, cochlear staining is restricted to IHCs and their supporting cells, with stronger expression in the apex than the base. By the same age, in the vestibular organs, NT3 expression is weak and restricted to saccular and utricular supporting cells. These results suggest that NT3 might play a long-term role in the maintenance and functioning of the adult auditory and vestibular systems and that supporting cells are the main source of this factor in the adult.

  View details for DOI 10.1002/cne.21227

  View details for PubMedID 17206617

• Studies of otic capsule morphology and gene expression in the Mov13 mouse - An animal model of type I osteogenesis imperfecta AUDIOLOGY AND NEURO-OTOLOGY Stankovic, K. M., Kristiansen, A. G., Bizaki, A., Lister, M., Adams, J. C., McKenna, M. J. 2007; 12 (5): 334-343

  Abstract

  Type I osteogenesis imperfecta (OI) is a disorder of skeletal bones characterized by bone fragility and blue sclera, which can result from mutations in genes encoding for type I collagen--the COL1A1 and COL1A2 genes. Fifty percent of patients with type I OI develop hearing loss and associated histopathological changes in the otic capsule that are indistinguishable from otosclerosis, a major cause of acquired hearing loss. In an attempt to elucidate molecular and cellular mechanisms of hearing loss in type I OI, we have studied the Mov13 mouse, which has served as an animal model of type I OI by virtue of exhibiting variable transcriptional block of the COL1A1 gene. We studied the morphometry of the Mov13 otic capsule and compared expression levels of 60 genes in the otic capsule with those in the tibia and parietal bone of the Mov13 and wild-type mice. The degree of transcriptional block of the COL1A1 gene and its downstream effects differed significantly between the bones examined. We found that expression levels of bone morphogenetic protein 3 and nuclear factor kappa-B1 best distinguished Mov13 otic capsule from wild-type otic capsule, and that osteoprotegerin, caspase recruitment domain containing protein 1, and partitioning defective protein 3 best distinguished Mov13 otic capsule from Mov13 tibia and parietal bone. Although the Mov13 mouse did not demonstrate evidence of active abnormal otic capsule remodeling as seen in type I OI and otosclerosis, studying gene expression in the Mov13 mouse has provided evidence that osteocytes of the otic capsule differ from osteocytes in other bones.

  View details for DOI 10.1159/000104789

  View details for Web of Science ID 000248238300007

  View details for PubMedID 17595534

• Current research in otosclerosis. Current opinion in otolaryngology & head and neck surgery Stankovic, K. M., McKenna, M. J. 2006; 14 (5): 347-51

  Abstract

  The aim of this article is to summarize and put into historical perspective current advances in research in otosclerosis, a disorder of the human temporal bone with a hereditary predisposition that is among the most common causes of acquired hearing loss.Genetic studies have revealed that otosclerosis is heterogeneous, with evidence for defects in at least seven genes associated with six distinct chromosomal loci. Measurements of high levels of osteoprotegerin expression in the normal otic capsule and soft tissues of the cochlea provide the first molecular insight as to why the normal otic capsule remodels minimally, if at all. Osteoprotegerin knockout mice provide the best available animal model to date to study abnormal otic capsule remodeling that closely resembles otosclerosis. There is mounting evidence that the measles virus plays an important role in pathogenesis of otosclerosis although the mechanisms by which the virus results in otosclerosis remain unknown. Quantitative measures of angiogenesis can reliably distinguish between clinical and histological otosclerosis. Advances in the emerging field of osteoimmunology will likely impact and benefit from the research in otosclerosis.Insights into molecular mechanisms that inhibit extensive remodeling in the normal otic capsule, and understanding of how these mechanisms are dysregulated in otosclerosis will allow future design of rational treatment strategies for otosclerosis.

  View details for DOI 10.1097/01.moo.0000244194.97301.19

  View details for PubMedID 16974150

• Survival of adult spiral ganglion neurons requires erbB receptor signaling in the inner ear JOURNAL OF NEUROSCIENCE Stankovic, K., Rio, C., Xia, A. P., Sugawara, M., Adams, J. C., Liberman, M. C., Corfas, G. 2004; 24 (40): 8651-8661

  Abstract

  Degeneration of cochlear sensory neurons is an important cause of hearing loss, but the mechanisms that maintain the survival of adult cochlear sensory neurons are not clearly defined. We now provide evidence implicating the neuregulin (NRG)-erbB receptor signaling pathway in this process. We found that NRG1 is expressed by spiral ganglion neurons (SGNs), whereas erbB2 and erbB3 are expressed by supporting cells of the organ of Corti, suggesting that these molecules mediate interactions between these cells. Transgenic mice in which erbB signaling in adult supporting cells is disrupted by expression of a dominant-negative erbB receptor show severe hearing loss and 80% postnatal loss of type-I SGNs without concomitant loss of the sensory cells that they contact. Quantitative RT-PCR analysis of neurotrophic factor expression shows a specific downregulation in expression of neurotrophin-3 (NT3) in the transgenic cochleas before the onset of neuronal death. Because NT3 is critical for survival of type I SGNs during development, these results suggest that it plays similar roles in the adult. Together, the data indicate that adult cochlear supporting cells provide critical trophic support to the neurons, that survival of postnatal cochlear sensory neurons depends on reciprocal interactions between neurons and supporting cells, and that these interactions are mediated by NRG and neurotrophins.

  View details for DOI 10.1523/JNEUROSCI.0733-04.2004

  View details for Web of Science ID 000224304100002

  View details for PubMedID 15470130

  View details for PubMedCentralID PMC6729966

• Real-time quantitative RT-PCR for low-abundance transcripts in the inner ear: analysis of neurotrophic factor expression HEARING RESEARCH Stankovic, K. M., Corfas, G. 2003; 185 (1-2): 97-108

  Abstract

  Real-time quantitative reverse transcription-PCR is a highly sensitive technology that allows high throughput quantification of gene expression. Application of this technique to the inner ear is potentially very important, but is not straightforward because tissue harvesting can be challenging, RNA yield from individual inner ears is low, and cDNA synthesis from scant RNA can be inefficient. To overcome these challenges, we tested many parameters and reagents, and developed an approach to reliably quantitate small changes in low-abundance transcripts. Using this technique we demonstrate the presence and quantify amounts of the neurotrophic factors neurotrophin 3 (NT-3), brain-derived neurotrophic factor (BDNF) and glial cell-line-derived neurotrophic factor (GDNF), in the cochlea and vestibular end organs of postnatal murine inner ear (P26). We show that out of the factors tested, BDNF is the only one differentially expressed between the cochlea and vestibular end organs, being 23.4+/-0.3 times more abundant in the vestibular end organs. Within the cochlea, GDNF gene expression is 4.9+/-0.2 times greater than NT-3 expression. Within the combined vestibular end organs, BDNF expression is 43.0+/-1.5 times greater than NT-3 expression. Our results suggest that neurotrophic factors continue to play a role in the postnatal inner ear, in addition to their previously shown essential role during development.

  View details for DOI 10.1016/S0378-5955(03)00298-3

  View details for Web of Science ID 000186562200011

  View details for PubMedID 14599697

• Medial efferent effects on auditory-nerve responses to tail-frequency tones II: Alteration of phase JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Stankovic, K. M., Guinan, J. J. 2000; 108 (2): 664-678

  Abstract

  It is often assumed that at frequencies in the tuning-curve tail there is a passive, constant coupling of basilar-membrane motion to inner hair cell (IHC) stereocilia. This paper shows changes in the phase of auditory-nerve-fiber (ANF) responses to tail-frequency tones and calls into question whether basilar-membrane-to-IHC coupling is constant. In cat ANFs with characteristic frequencies > or = 10 kHz, efferent effects on the phase of ANF responses to tail-frequency tones were measured. Efferent stimulation caused substantial changes in ANF phase (deltaphi) (range -80 degrees to +60 degrees, average -15 degrees, a phase lag) with the largest changes at sound levels near threshold and 3-4 octaves below characteristic frequency (CF). At these tail frequencies, efferent stimulation had much less effect on the phase of the cochlear microphonic (CM) than on ANF phase. Thus, since CM is synchronous with basilar-membrane motion for low-frequency stimuli in the cochlear base, the efferent-induced change in ANF phase is unlikely to be due entirely to a change in basilar-membrane phase. At tail frequencies, ANF phase changed with sound level (often by 90 degrees-180 degrees) and the deltaphi from a fiber was positively correlated with the slope of its phase-versus-sound-level function at the same frequency, as if deltaphi were caused by a 2-4 dB increase in sound level. This correlation suggests that the processes that produce the change in ANF phase with sound level at tail frequencies are also involved in producing deltaphi. It is hypothesized that both efferent stimulation and increases in sound level produce similar phase changes because they both produce a similar mix of cochlear vibrational modes.

  View details for DOI 10.1121/1.429599

  View details for Web of Science ID 000088626300026

  View details for PubMedID 10955633

• Medial efferent effects on auditory-nerve responses to tail-frequency tones. I. Rate reduction JOURNAL OF THE ACOUSTICAL SOCIETY OF AMERICA Stankovic, J. M., Guinan, J. J. 1999; 106 (2): 857-869

  Abstract

  One way medial efferents are thought to inhibit responses of auditory-nerve fibers (ANFs) is by reducing the gain of the cochlear amplifier thereby reducing motion of the basilar membrane. If this is the only mechanism of medial efferent inhibition, then medial efferents would not be expected to inhibit responses where the cochlear amplifier has little effect, i.e., at sound frequencies in the tails of tuning curves. Inhibition at tail frequencies was tested for by obtaining randomized rate-level functions from cat ANFs with high characteristic frequencies (CF > or = 5 kHz), stimulated with tones two or more octaves below CF. It was found that electrical stimulation of medial efferents can indeed inhibit ANF responses to tail-frequency tones. The amplitude of efferent inhibition depended on both sound level (largest near to threshold) and frequency (largest two to three octaves below CF). On average, inhibition of high-CF ANFs responding to 1 kHz tones was around 5 dB. Although an efferent reduction of basilar-membrane motion cannot be ruled out as the mechanism producing the inhibition of ANF responses to tail frequency tones, it seems more likely that efferents produce this effect by changing the micromechanics of the cochlear partition.

  View details for DOI 10.1121/1.427102

  View details for Web of Science ID 000081993700036

  View details for PubMedID 10462791

• Localization of pH regulating proteins H+ATPase and Cl-/HCO3- exchanger in the guinea pig inner ear. Hearing research Stanković, K. M., Brown, D., Alper, S. L., Adams, J. C. 1997; 114 (1-2): 21-34

  Abstract

  Mechanisms that regulate endolymphatic pH are unknown. It has long been recognized that, because of the large positive endolymphatic potential in the cochlea, a passive movement of protons would be directed out of endolymph leading to endolymphatic alkalization. However, endolymphatic pH is close to that of blood, suggesting that H+ is being secreted into endolymph. Since the kidney and the inner ear are both actively engaged in fluid and electrolyte regulation, we attempted to determine whether proteins responsible for acid secretion in the kidney also exist in the guinea pig inner ear. To that end, a monoclonal antibody against a 31 kDa subunit of a vacuolar vH+ATPase and a polyclonal, affinity purified antibody against the AE2 Cl-/HCO3- exchanger (which can also recognize AE1 under some conditions) were used. In the cochlea, the strongest immunoreactivity for the vH+ATPase was found in apical plasma membranes and apical cytoplasm of strial marginal cells. These cells were negative for the Cl-/HCO3- exchanger. Certain cells of the inner ear demonstrated both apical staining for vH+ATPase and basolateral staining for the Cl-/HCO3- exchanger; these included interdental cells and epithelial cells of the endolymphatic sac. Cochlear cell types with diffuse cytoplasmic staining for vH+ATPase and a basolaterally localized Cl-/HCO3- exchanger included inner hair cells, root cells and a subset of supporting cells in the organ of Corti. Hair cells of the utricle, saccule and cristae ampullaris also expressed both vH+ATPase and the Cl-/HCO3- exchanger, but immunostaining for the vH+ATPase was less intense and less polarized than in the cochlea. These immunocytochemical results support a role for the vH+ATPase and Cl-/HCO3- exchanger in the regulation of endolymphatic pH and suggest that certain cells (including strial marginal cells and epithelial cells of the endolymphatic sac) may be specialized for this regulation.

  View details for DOI 10.1016/s0378-5955(97)00072-5

  View details for PubMedID 9447915

• Medial efferent inhibition produces the largest equivalent attenuations at moderate to high sound levels in cat auditory-nerve fibers. The Journal of the Acoustical Society of America Guinan, J. J., Stankovic, K. M. 1996; 100 (3): 1680-90

  Abstract

  Previous work has shown that medial efferents can inhibit responses of auditory-nerve fibers to high-level sounds and that fibers with low spontaneous rates (SRs) are inhibited most. However, quantitative interpretation of these data is made difficult by effects of adaptation. To minimize systematic differences in adaptation, efferent inhibition was measured with a randomized presentation of both sound level and efferent stimulation. In anesthetized cats, efferents were stimulated with 200/s shocks and auditory-nerve-fiber responses were recorded for tone bursts (0-100 dB SPL, 5-dB steps) at their characteristic frequencies. Below 50 dB SPL, efferent inhibition (measured as equivalent attenuation) was similar for all fibers with similar CFs in the same cat. At 45-75 dB SPL, low-SR and medium-SR fibers often showed much larger inhibition, and substantial inhibition even at 100 dB SPL. Expressed as a fractional decrease in rate, at 90-100 dB SPL the inhibition was 0%, 6%, and 13% for high-, medium-, and low-SR fibers (differences statistically significant). Finding the largest equivalent attenuations at 45-75 dB SPL does not fit with the hypothesis that medial-efferent inhibition is due solely to a reduction of basilar-membrane motion. The large attenuations, some over 50 dB, indicate that medial efferent inhibition is more potent than previously reported.

  View details for DOI 10.1121/1.416066

  View details for PubMedID 8817894

• Immunolocalization of aquaporin CHIP in the guinea pig inner ear. The American journal of physiology Stanković, K. M., Adams, J. C., Brown, D. 1995; 269 (6 Pt 1): C1450-6

  Abstract

  Aquaporin CHIP (AQP-CHIP) is a water channel protein previously identified in red blood cells and water transporting epithelia. The inner ear is an organ of hearing and balance whose normal function depends critically on maintenance of fluid homeostasis. In this study, AQP-CHIP, or a close homologue, was found in specific cells of the inner ear, as assessed by immunocytochemistry with the use of affinity-purified polyclonal antibodies against AQP-CHIP.AQP-CHIP was predominantly found in fibrocytes in close association with bone, including most of the cells lining the bony labyrinth and in fibrocytes lining the endolymphatic duct and sac. AQP-CHIP-positive cells not directly apposing bone include cells under the basilar membrane, some type III fibrocytes of the spiral ligament, fibrocytes of the spiral limbus, and the trabecular perilymphatic tissue extending from the membranous to the bony labyrinth. AQP-CHIP was also found in the periosteum of the middle ear and cranial bones, as well as in chondrocytes of the oval window and stapes. The distribution of AQP-CHIP in the inner ear suggests that AQP-CHIP may have special significance for maintenance of bone and the basilar membrane, and for function of the spiral ligament.

  View details for DOI 10.1152/ajpcell.1995.269.6.C1450

  View details for PubMedID 8572173