Cochlear organoids reveal transcriptional programs of postnatal hair cell differentiation from supporting cells.

We explore the changes in chromatin accessibility and transcriptional programs for cochlear hair cell differentiation from postmitotic supporting cells using organoids from postnatal cochlea. The organoids contain cells with transcriptional signatures of differentiating vestibular and cochlear hair cells. Construction of trajectories identifies Lgr5+ cells as progenitors for hair cells, and the genomic data reveal gene regulatory networks leading to hair cells. We validate these networks, demonstrating dynamic changes both in expression and predicted binding sites of transcription factors (TFs) during organoid differentiation. We identify known regulators of hair cell development, Atoh1, Pou4f3, and Gfi1, and the analysis predicts the regulatory factors Tcf4, an E-protein and heterodimerization partner of Atoh1, and Ddit3, a CCAAT/enhancer-binding protein (C/EBP) that represses Hes1 and activates transcription of Wnt-signaling-related genes. Deciphering the signals for hair cell regeneration from mammalian cochlear supporting cells reveals candidates for hair cell (HC) regeneration, which is limited in the adult.

HIC1 Represses Atoh1 Transcription and Hair Cell Differentiation in the Cochlea.

Across species, expression of the basic helix-loop-helix transcription factor ATOH1 promotes differentiation of cochlear supporting cells to sensory hair cells required for hearing. In mammals, this process is limited to development, whereas nonmammalian vertebrates can also regenerate hair cells after injury. The mechanistic basis for this difference is not fully understood. Hypermethylated in cancer 1 (HIC1) is a transcriptional repressor known to inhibit Atoh1 in the cerebellum. We therefore investigated its potential role in cochlear hair cell differentiation. We find that Hic1 is expressed throughout the postnatal murine cochlear sensory epithelium. In cochlear organoids, Hic1 knockdown induces Atoh1 expression and promotes hair cell differentiation, while Hic1 overexpression hinders differentiation. Wild-type HIC1, but not the DNA-binding mutant C521S, suppresses activity of the Atoh1 autoregulatory enhancer and blocks its responsiveness to ß-catenin activation. Our findings reveal the importance of HIC1 repression of Atoh1 in the cochlea, which may be targeted to promote hair cell regeneration.

Considerations in Management of Acute Otitis Media in the COVID-19 Era.

OBJECTIVES: To present the otologic findings of a patient with COVID-19 and complicated acute otitis media, evaluate for the presence of SARS-CoV-2 in middle ear fluid, and assess whether suctioning of middle ear fluid may be aerosol- generating. METHODS: The case of a man with SARS-CoV-2 infection and complicated acute otitis media with facial paralysis is presented to illustrate unique clinical decisions made in context of the COVID-19 pandemic. A cadaveric temporal bone was used to simulate droplet spread during suctioning of fluorescein-labelled middle ear fluid and visualized with a blue-light filter. RESULTS: A 23-year-old male who presented with complicated acute otitis media with facial paralysis was found to have an acute infection with SARS-CoV-2, with positive viral PCR of nasopharyngeal swab, and a negative PCR of the middle ear fluid. He was placed on isolation precautions and treated with myringotomy, topical and systemic antibiotics, and antivirals. Consistent with observations during endonasal suctioning, suctioning of middle ear fluid was not found to be aerosol or droplet generating. CONCLUSION: The case of a patient with active COVID-19 presenting with complicated acute otitis media in whom middle ear fluid was sampled to evaluate the etiology of the infection and the potential middle ear predilection of SARS-CoV-2 is described. This study has implications for the clinical management of patients with both known and unknown SARS-CoV-2 infection who present with ear disease. While middle ear suctioning may not be aerosol-generating, the risk of coughing or prolonged close contact requires heightened precautions during otologic procedures in patients with suspected or confirmed COVID-19.

Aerosol Dispersion During Mastoidectomy and Custom Mitigation Strategies for Otologic Surgery in the COVID-19 Era.

OBJECTIVE: To investigate small-particle aerosolization from mastoidectomy relevant to potential viral transmission and to test source-control mitigation strategies. STUDY DESIGN: Cadaveric simulation. SETTING: Surgical simulation laboratory. METHODS: An optical particle size spectrometer was used to quantify 1- to 10-µm aerosols 30 cm from mastoid cortex drilling. Two barrier drapes were evaluated: OtoTent1, a drape sheet affixed to the microscope; OtoTent2, a custom-structured drape that enclosed the surgical field with specialized ports. RESULTS: Mastoid drilling without a barrier drape, with or without an aerosol-scavenging second suction, generated large amounts of 1- to 10-µm particulate. Drilling under OtoTent1 generated a high density of particles when compared with baseline environmental levels (P < .001, U = 107). By contrast, when drilling was conducted under OtoTent2, mean particle density remained at baseline. Adding a second suction inside OtoTent1 or OtoTent2 kept particle density at baseline levels. Significant aerosols were released upon removal of OtoTent1 or OtoTent2 despite a 60-second pause before drape removal after drilling (P < .001, U = 0, n = 10, 12; P < .001, U = 2, n = 12, 12, respectively). However, particle density did not increase above baseline when a second suction and a pause before removal were both employed. CONCLUSIONS: Mastoidectomy without a barrier, even when a second suction was added, generated substantial 1- to 10-µm aerosols. During drilling, large amounts of aerosols above baseline levels were detected with OtoTent1 but not OtoTent2. For both drapes, a second suction was an effective mitigation strategy during drilling. Last, the combination of a second suction and a pause before removal prevented aerosol escape during the removal of either drape.

Topical fibroblast growth factor-2 for treatment of chronic tympanic membrane perforations.

OBJECTIVE: To determine the efficacy of fibroblast growth factor-2 (FGF-2) in treating chronic nonhealing tympanic membrane (TM) perforations. METHOD: 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. RESULTS: 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. CONCLUSION: 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. LEVEL OF EVIDENCE: 1b.

Transmastoid Labyrinthectomy for Menière's Disease: Experience and Outcomes.

OBJECTIVE: To characterize presurgical symptoms and treatment history and postoperative course in patients with medically recalcitrant Menière's disease undergoing transmastoid labyrinthectomy in the post-intratympanic gentamicin era. STUDY DESIGN: Retrospective case series. SETTING: Tertiary academic medical center. PATIENTS: All patients who underwent transmastoid labyrinthectomy for medically recalcitrant Menière's disease in 2003 to 2019 by the senior author. INTERVENTIONS: Review of patients' medical records for: preoperative history of drop attacks, gentamicin injections, endolymphatic sac decompression or vestibular neurectomy, preoperative audiograms, length of hospital stay, postoperative complications, and persistent symptoms or challenging recovery. MAIN OUTCOME MEASURES: Presurgical clinical history and proximal postoperative outcomes. RESULTS: Seventy-two patients with a mean age of 56.7 (standard deviation [SD] 10.7) were included. All cases were unilateral. Forty-three patients (59.7%) suffered from drop attacks. Sixty-two (86.1%) had failed sufficient symptom control with gentamicin injections. The mean preoperative word recognition score was 36.4% (SD 23.7) versus 95.1% (SD 8.5) in the contralateral ear. The mean pure-tone average (PTA) of the ipsilateral ear before surgery was 65.5 dB (SD 18.0) versus 16.2 (SD 13.5) for the contralateral ear. Mean hospital stay was 2.0 days (SD 0.87 days, range of 1-5 d). Three patients (4.2%) had prolonged postoperative vertigo. CONCLUSIONS: Transmastoid labyrinthectomy at our center is performed for unilateral Menière's disease, generally when intratympanic gentamicin has failed. A majority of surgical patients suffer from drop attacks preoperatively. Hospital stay is typically brief.

Applications of Lgr5-Positive Cochlear Progenitors (LCPs) to the Study of Hair Cell Differentiation.

The mouse cochlea contains approximately 15,000 hair cells. Its dimensions and location, and the small number of hair cells, make mechanistic, developmental and cellular replacement studies difficult. We recently published a protocol to expand and differentiate murine neonatal cochlear progenitor cells into 3D organoids that recapitulate developmental pathways and can generate large numbers of hair cells with intact stereociliary bundles, molecular markers of the native cells and mechanotransduction channel activity, as indicated by FM1-43 uptake. Here, we elaborate on the method and application of these Lgr5-positive cochlear progenitors, termed LCPs, to the study of inner ear development and differentiation. We demonstrate the use of these cells for testing several drug candidates, gene silencing and overexpression, as well as genomic modification using CRISPR/Cas9. We thus establish LCPs as a valuable in vitro tool for the analysis of progenitor cell manipulation and hair cell differentiation.

The Difficult Secondary Tracheoesophageal Puncture: A Technique for Safe Insertion.

BACKGROUND/AIMS: The tracheoesophageal prothesis (TEP) has become the primary modality for laryngeal communication after total laryngectomy due to high success rates, minimal morbidity, and more natural pulmonary driven speech. Fibrosis, kyphosis, and post-radiation contracture may preclude TEP placement through rigid esophagoscopy, and certain patients may not tolerate an in-office awake procedure. For such patients, a technique for flexible esophageal stenting and TEP placement is necessary. METHODS: We performed a retrospective review of 3 patients who underwent TEP placement through endotracheal-tube esophageal stenting at the Massachusetts Eye and Ear Infirmary. RESULTS: All 3 patients underwent laryngectomy after prior chemoradiotherapy for laryngeal cancer with resulting neck contracture and fibrosis preventing rigid esophagoscopy. All patients underwent successful TEP placement through endotracheal stenting without complication and developed excellent tracheoesophageal speech. Specific technical details are highlighted. CONCLUSIONS: In patients with anatomical constraints preventing traditional TEP placement through rigid esophagoscopy, fiberoptic guidance through an endotracheal tube stent provides a safe and efficient approach for TEP placement.

ERBB2 signaling drives supporting cell proliferation in vitro and apparent supernumerary hair cell formation in vivo in the neonatal mouse cochlea.

In mammals, cochlear hair cells are not regenerated once they are lost, leading to permanent hearing deficits. In other vertebrates, the adjacent supporting cells act as a stem cell compartment, in that they both proliferate and differentiate into de novo auditory hair cells. Although there is evidence that mammalian cochlear supporting cells can differentiate into new hair cells, the signals that regulate this process are poorly characterized. We hypothesize that signaling from the epidermal growth factor receptor (EGFR) family may play a role in cochlear regeneration. We focus on one such member, ERBB2, and report the effects of expressing a constitutively active ERBB2 receptor in neonatal mouse cochlear supporting cells, using viruses and transgenic expression. Lineage tracing with fluorescent reporter proteins was used to determine the relationships between cells with active ERBB2 signaling and cells that divided or differentiated into hair cells. In vitro, individual supporting cells harbouring a constitutively active ERBB2 receptor appeared to signal to their neighbouring supporting cells, inducing them to down-regulate a supporting cell marker and to proliferate. In vivo, we found supernumerary hair cell-like cells near supporting cells that expressed ERBB2 receptors. Both supporting cell proliferation and hair cell differentiation were largely reproduced in vitro using small molecules that we show also activate ERBB2. Our data suggest that signaling from the receptor tyrosine kinase ERBB2 can drive the activation of secondary signaling pathways to regulate regeneration, suggesting a new model where an interplay of cell signaling regulates regeneration by endogenous stem-like cells.

Temporal bone computed tomography findings associated with feasibility of endoscopic ear surgery.

PURPOSE: There are no formal radiologic criteria to stratify patients for transcanal (TEES) or transmastoid endoscopic ear surgery for resection of cholesteatoma. We aim to determine 1) whether standard preoperative computed tomography (CT) findings are associated with the need for conversion to a transmastoid approach and 2) the amount of time added for conversion from TEES to transmastoid techniques. MATERIALS AND METHODS: Retrospective chart review of consecutive pediatric and adult cases of TEES for primary cholesteatoma from 2013 through 2015 (n=52). TEES cases were defined as endoscope-only procedures that did not require a transmastoid approach (n=33). Conversion cases were defined as procedures that began as TEES however, required conversion to a transmastoid approach due to the inability to complete cholesteatoma removal (n=19). Preoperative CT findings and total operating room (OR) times of TEES and conversion cases were compared. RESULTS: Preoperative CT scan characteristics that were associated with conversion included tegmen erosion (p=0.026), malleus erosion (p<0.001), incus erosion (p=0.009), mastoid opacification (p=0.009), soft tissue opacification extending into the aditus ad antrum (p=0.009) and into antrum (p=0.006). Total OR time for TEES cases was significantly shorter than conversion cases (median 143min versus 217min, p<0.001). CONCLUSIONS: Preoperative CT findings, notably extension of soft tissue in the aditus ad antrum, antrum and mastoid, are associated with need for conversion to transmastoid technique to achieve removal of cholesteatoma. Endoscope-only cases were significantly faster than cases that required conversion to a transmastoid approach.

Global health-related publications in otolaryngology are increasing.

OBJECTIVES/HYPOTHESIS: Determine trends in global health-related publication in otolaryngology. STUDY DESIGN: A review of research databases. METHODS: A search of publications available on PubMed and nine additional databases was undertaken reviewing two time periods 10 years apart for the timeframes 1998 to 2002 (early time period) and 2008 to 2012 (recent time period) using specific search terms to identify global health-related publications in otolaryngology. Publications were examined for region of origin, subspecialty, type of publication, and evidence of international collaboration. χ and t test analyses were used to identify trends. RESULTS: In the 1998 to 2002 time period, a total of 26 publications met inclusion criteria for the study, with a mean of 5.2 ± 2.8 publications per year. In the 2008 to 2012 time period, a total of 61 publications met inclusion criteria, with a mean of 12.3 ± 5.6 publications per year. The 235% increase in global health-related publications identified between the two study periods was statistically significant (P = .02). The absolute number of publications in which collaboration occurred between countries increased from three in the early time period to nine the recent time period. CONCLUSIONS: There has been a significant increase in the volume of global health-related publications in English language otolaryngology journals over the past decade, providing strong evidence of the increasing trend of global health as an academic pursuit within the field of otolaryngology.

Surgical management of cervical chyloma following parathyroidectomy.

Although rare, chylomas can present as a neck mass, especially in the post-operative setting. Here, we present a case of a persistent cervical chyloma following parathyroidectomy and propose a management algorithm for this clinical entity.

Folding and binding integrity of variants of a prototype ligand-binding module from the LDL receptor possessing multiple alanine substitutions.

The LA repeats that comprise the ligand-binding domain of the LDL receptor are among the most common autonomously structured extracellular modules found in the nonredundant protein sequence database. Here, we investigate the information content of the amino acid sequence of a typical LA module by constructing sequences with alanine residues at nonconserved positions in the module. Starting with the sequence of the fifth ligand-binding repeat of the LDL receptor (LA5), we created generic LA modules with alanine substitutions of nonconserved residues in only the N-terminal lobe, only the C-terminal lobe, and throughout both lobes of the module. LA variants with alanine residues at as many as 18 of 37 positions fold to a preferred disulfide isomer in the presence of calcium. Indeed, the six cysteines, the C-terminal calcium coordinating residues, two hydrophobic residues involved in packing, two glycines, and five other residues that form side chain-intramodule hydrogen bonds are alone sufficient to specify the fold of an LA module when alanine residues are present at all other positions. The LA variants with multiple alanines in either the N- or C-terminal lobe were then exploited to identify residues of LA5 that contribute to the binding of apoE-containing ligands in LDL receptor-derived "minireceptors", implicating nonconserved residues of the N-terminal lobe of LA5 in recognition of apoE-DMPC. Our library of LA modules with multiple alanine substitutions should be generally useful for probing the roles of nonconserved side chains in ligand recognition by proteins of the LDL receptor family.

A two-module region of the low-density lipoprotein receptor sufficient for formation of complexes with apolipoprotein E ligands.

The low-density lipoprotein (LDL) receptor transports two different classes of cholesterol-carrying lipoprotein particles into cells: LDL particles, which contain a single copy of apolipoprotein B-100 (apoB-100), and beta-migrating very low-density lipoprotein (beta-VLDL) particles, which contain multiple copies of apolipoprotein E (apoE). The ligand-binding domain of the receptor lies at its amino-terminal end within seven adjacent LDL-A repeats (LA1-LA7). Although prior work clearly establishes that LA5 is required for high-affinity binding of particles containing apolipoprotein E (apoE), the number of ligand-binding repeats sufficient to bind apoE ligands has not yet been determined. Similarly, uncertainty exists as to whether a single lipid-activated apoE receptor-binding site within a particle is capable of binding to the LDLR with high affinity or whether more than one is required. Here, we establish that the LA4-5 two-repeat pair is sufficient to bind apoE-containing ligands, on the basis of binding studies performed with a series of LDLR-derived "minireceptors" containing up to four repeats. Using single chain multimers of the apoE receptor-binding domain (N-apoE), we also show that more than one receptor-binding site in its lipid-activated conformation is required to bind to the LDLR with high affinity. Thus, in addition to inducing a conformational change in the structure of N-apoE, lipid association enhances the affinity of apoE for the LDLR in part by creating a multivalent ligand.