Corrigendum: Characterizing Adult Cochlear Supporting Cell Transcriptional Diversity Using Single-Cell RNA-Seq: Validation in the Adult Mouse and Translational Implications for the Adult Human Cochlea.

[This corrects the article DOI: 10.3389/fnmol.2020.00013.].

Characterizing Adult Cochlear Supporting Cell Transcriptional Diversity Using Single-Cell RNA-Seq: Validation in the Adult Mouse and Translational Implications for the Adult Human Cochlea.

Hearing loss is a problem that impacts a significant proportion of the adult population. Cochlear hair cell (HC) loss due to loud noise, chemotherapy and aging is the major underlying cause. A significant proportion of these individuals are dissatisfied with available treatment options which include hearing aids and cochlear implants. An alternative approach to restore hearing would be to regenerate HCs. Such therapy would require a recapitulation of the complex architecture of the organ of Corti, necessitating regeneration of both mature HCs and supporting cells (SCs). Transcriptional profiles of the mature cell types in the cochlea are necessary to can provide a metric for eventual regeneration therapies. To assist in this effort, we sought to provide the first single-cell characterization of the adult cochlear SC transcriptome. We performed single-cell RNA-Seq on FACS-purified adult cochlear SCs from the LfngEGFP adult mouse, in which SCs express GFP. We demonstrate that adult cochlear SCs are transcriptionally distinct from their perinatal counterparts. We establish cell-type-specific adult cochlear SC transcriptome profiles, and we validate these expression profiles through a combination of both fluorescent immunohistochemistry and in situ hybridization co-localization and quantitative polymerase chain reaction (qPCR) of adult cochlear SCs. Furthermore, we demonstrate the relevance of these profiles to the adult human cochlea through immunofluorescent human temporal bone histopathology. Finally, we demonstrate cell cycle regulator expression in adult SCs and perform pathway analyses to identify potential mechanisms for facilitating mitotic regeneration (cell proliferation, differentiation, and eventually regeneration) in the adult mammalian cochlea. Our findings demonstrate the importance of characterizing mature as opposed to perinatal SCs.

Temporal Bone Histopathology of First-Generation Cochlear Implant Electrode Translocation.

OBJECTIVE: To evaluate the histopathology of human temporal bones (HTBs) with cochlear implants (CI). BACKGROUND: Understanding CI translocation injuries is critical for improving outcomes. MATERIAL AND METHODS: Thirteen HTBs from 12 CI patients were studied. Six HTBs exhibited translocation with localized injury (Group 1) and seven HTBs exhibited translocation with significant lateral wall injury (Group 2). There were no significant differences between Group 1 and Group 2 for age at death, age at implantation, and years with CI. RESULTS: Four out of six of Group 1 had round window approach, while all seven of Group 2 had cochleostomy approach. Translocation injuries tended to occur near 180 degrees of angular insertion with a mean of 186.36 ±â€Š51.62 degrees. Average CI insertion length for Group 2 was 21.86 ±â€Š2.55 mm, significantly longer than Group 1 at 18.50 ±â€Š3.33 mm (p = 0.031). Group 1 had an average of 17300 ±â€Š9415 spiral ganglia neurons (SGNs) while Group 2 had significantly fewer SGNs 6714 ±â€Š4269 (p = 0.015). Group 1 average auditory performance of 66.55 ±â€Š27.20% was higher than that of Group 2 of 39.86 ±â€Š15.36%. Group 2 had a high degree of osteoneogenesis and infiltration of cells generally localized to areas of translocation injury and cochleostomy. CONCLUSION: Translocation injuries tend to occur at an insertion angle of 180 degrees, at 9 to 10 mm. Lateral wall injury and damage to the organ of Corti incites fibrosis, osteoneogenesis, and infiltration, lower SGN count and poorer auditory performance. Longer electrodes were more prone to translocation and higher chance of significant intracochlear injury.

A Histopathologic Comparison of Eustachian Tube Anatomy in Pediatric and Adult Temporal Bones.

HYPOTHESIS: In children, the distance between the carotid canal (CC) and Eustachian tube (ET) is not significantly narrower than the adult population. BACKGROUND: ET dysfunction treated with ET dilation is FDA approved for adults. Several studies describe the close relationship between the CC and the ET in adults, but the anatomy of the ET has not been well defined in children. This study seeks to investigate these relationships in the pediatric population. METHODS: Histologic sections from 23 temporal bones of pediatric patients ages 0 to 18 were reviewed by two independent observers. The distance between the CC and the cartilaginous Eustachian tube (CET), bony-cartilaginous junction (BCJ), and bony Eustachian tube (BET) were measured. Fifteen adult temporal bones were used as a control group. RESULTS: The distance to the CC was narrowest at the BET, and was actually higher in the pediatric population when compared to adults (0.5 mm and 0.2 mm, respectively, p = 0.06). The CC-CET distance was smaller in the pediatric group (2.3 mm vs 3.3 mm, p < 0.01). The bony-cartilaginous junction is often the region of most concern during dilation. There was no significant difference between the CC-BET distance in pediatric and adult groups (1.9 vs 2.3 mm, p = 0.20). CONCLUSIONS: CET-CC is smaller in the pediatric population, as expected due to smaller anatomic structures. However, the variable incline of the ET results in a CC-BCJ distance that is similar to the adult population. Although imaging studies are necessary.

Supporting cell survival after cochlear implant surgery.

Supporting cells (SCs) provide structure and maintain an environment that allows hair cells to receive and transmit signals in the auditory pathway. After insult to hair cells and ganglion cells, SCs respond by marking unsalvageable cells for death and maintain structural integrity. Although the histopathology after cochlear implantation has been described regarding hair cells and neural structures, surviving SCs in the implanted ear have not. We present a patient whose posthumous examination of an implanted cochlea demonstrated SC survival. This finding has implications for SC function in maintaining electrical hearing and candidacy for future hair cell regeneration therapies. Laryngoscope, 129:E36-E40, 2019.

Connexin 26 Immunohistochemistry in Temporal Bones With Cochlear Otosclerosis.

HYPOTHESIS: Connexin-26 (Cx26) expression is diminished in the spiral ligament of subjects with hearing loss and cochlear otosclerosis (CO). BACKGROUND: Human temporal bone (HTB) studies have demonstrated that CO is associated with hyalinization of the spiral ligament. We hypothesize that hyalinization is associated with a loss of fibrocytes with a consequent decline in Cx26 expression. Cx26 and Connexin-30 (Cx30) encode gap junction proteins expressed in supporting cells of the organ of Corti, the spiral limbus, stria vascularis, and in fibrocytes of the spiral ligament. These gap junctions are critical for potassium recycling and maintenance of the endocochlear potential. Diminished expression of these proteins would likely be associated with hearing dysfunction. METHODS: Histopathology and clinical characteristics of 45 HTB specimens with CO and spiral ligament hyalinization were reviewed. Those with sensorineural or mixed hearing loss but normal or near-normal hair cell counts were analyzed with light microscopy, and Cx26-immunoreactive (IR) signal was qualitatively assessed. RESULTS: H&E staining demonstrated hyalinization in the spiral ligament and loss of type II and type III fibrocytes. Cx26-IR was diminished throughout the cochlea affected with CO compared with normal controls. CONCLUSIONS: Cx26-IR reduction in the spiral ligament of subjects with CO likely plays a role in hearing loss.

Cochlear Dysfunction is not Common in Human Meningioma of the Internal Auditory Canal.

HYPOTHESIS: Cochlear dysfunction is not common in human meningioma of the internal auditory canal. BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Cochlear implant histopathology.

The microscopic examination of fifty-five serially sectioned implanted temporal bones has provided insight into what is being stimulated; and the changes that are the result of the insertion and presence of the implant. The ganglion cell bodies (neurons) are structures being stimulated (two laboratories have reported an inverse relationship of the number of neurons and performance). Insertion through the round window, verses a cochleostomy, produces the least fibrosis and new bone. Fibrosis and new bone do not affect the implant function unless they form in the scala vestibuli in the region of the ductus reuniens, and, block it; and produce cochlear hydrops resulting in a delayed low tone loss of hearing in hybrid implants. Animal models cannot be applied to humans because of the difference in size and myelination of the neurons.

Post Hybrid Cochlear Implant Hearing Loss and Endolymphatic Hydrops.

OBJECTIVE: To evaluate for potential causes of delayed loss of residual hearing that variably occurs with hybrid cochlear implants. STUDY DESIGN: Histopathological evaluation of 29 human temporal bone (HTB) with cochlear implant (CI). SETTING: The Neurotology and House HTB Laboratory of UCLA (House-UCLA). SUBJECTS AND METHODS: HTB from CI patients from the House-UCLA HTB Laboratory (n = 28) and one courtesy of Massachusetts Eye and Ear Infirmary (MEEI). Histopathological analysis to identify the location of cochleostomy, fibrosis, and bone formation in the scala vestibuli and tympani, and endolymphatic hydrops. Spiral ganglion neuron counts were obtained. Statistical analysis compared presence of cochleostomy and location with the histopathological findings. RESULTS: Seventeen of 29 bones with fibrosis in the scala vestibule (SV) and tympani had evidence of a cochleostomy involving the SV containing the ductus reunions, all of which had hydrops. Ten of 11 bones had no SV fibrosis, and a cochleostomy limited to the scala tympani, of which all had no hydrops. One HTB had moderate SV fibrosis not involving the ductus reuniens, and was without hydrops. One HTB had a SV cochleostomy but the electrode ruptured Reissner's membrane, and was without hydrops. Cochleostomy was significantly associated with SV fibrosis and hydrops (p < 0.01), those without hydrops had no SV atrophy (p < 0.01). Round window insertion was associated with no fibrosis and no hydrops. CONCLUSION: We hypothesize that cochleostomies involving scala vestibuli incite fibrosis, compromising the ductus reuniens, causing hydrops which may cause the delayed loss of residual low frequency hearing in CI.

Immunohistochemical techniques for the human inner ear.

In this review, we provide a description of the recent methods used for immunohistochemical staining of the human inner ear using formalin-fixed frozen, paraffin and celloidin-embedded sections. We also show the application of these immunohistochemical methods in auditory and vestibular endorgans microdissected from the human temporal bone. We compare the advantages and disadvantages of immunohistochemistry (IHC) in the different types of embedding media. IHC in frozen and paraffin-embedded sections yields a robust immunoreactive signal. Both frozen and paraffin sections would be the best alternative in the case where celloidin-embedding technique is not available. IHC in whole endorgans yields excellent results and can be used when desiring to detect regional variations of protein expression in the sensory epithelia. One advantage of microdissection is that the tissue is processed immediately and IHC can be made within 1 week of temporal bone collection. A second advantage of microdissection is the excellent preservation of both morphology and antigenicity. Using celloidin-embedded inner ear sections, we were able to detect several antigens by IHC and immunofluorescence using antigen retrieval methods. These techniques, previously applied only in animal models, allow for the study of numerous important proteins expressed in the human temporal bone potentially opening up a new field for future human inner ear research.

Empirical Derivation of Correction Factors for Human Spiral Ganglion Cell Nucleus and Nucleolus Count Units.

OBJECTIVE: Profile count method for estimating cell number in sectioned tissue applies a correction factor for double count (resulting from transection during sectioning) of count units selected to represent the cell. For human spiral ganglion cell counts, we attempted to address apparent confusion between published correction factors for nucleus and nucleolus count units that are identical despite the role of count unit diameter in a commonly used correction factor formula. STUDY DESIGN: We examined a portion of human cochlea to empirically derive correction factors for the 2 count units, using 3-dimensional reconstruction software to identify double counts. SETTING: The Neurotology and House Histological Temporal Bone Laboratory at University of California at Los Angeles. SUBJECTS AND METHODS: Using a fully sectioned and stained human temporal bone, we identified and generated digital images of sections of the modiolar region of the lower first turn of cochlea, identified count units with a light microscope, labeled them on corresponding digital sections, and used 3-dimensional reconstruction software to identify double-counted count units. RESULTS: For 25 consecutive sections, we determined that double-count correction factors for nucleus count unit (0.91) and nucleolus count unit (0.92) matched the published factors. We discovered that nuclei and, therefore, spiral ganglion cells were undercounted by 6.3% when using nucleolus count units. CONCLUSION: We determined that correction factors for count units must include an element for undercounting spiral ganglion cells as well as the double-count element. We recommend a correction factor of 0.91 for the nucleus count unit and 0.98 for the nucleolus count unit when using 20-µm sections.

Identification of target proteins involved in cochlear otosclerosis.

HYPOTHESIS: Investigation of differential protein expression will provide clues to pathophysiology in otosclerosis. BACKGROUND: Otosclerosis is a bone remodeling disorder limited to the endochondral layer of the otic capsule within the temporal bone. Some authors have suggested an inflammatory etiology for otosclerosis resulting from persistent measles virus infection involving the otic capsule. Despite numerous genetic studies, implication of candidate genes in the otosclerotic process remains elusive. We employed liquid chromatography-mass spectrometry (LC-MS) analysis on formalin-fixed celloidin-embedded temporal bone tissues for postmortem investigation of otosclerosis. METHODS: Proteomic analysis was performed using human temporal bones from a patient with severe otosclerosis and a control temporal bone. Sections were dissected under microscopy to remove otosclerotic lesions and normal otic capsule for proteomic analysis. Tandem 2D chromatography mass spectrometry was employed. Data analysis and peptide matching to FASTA human databases was done using SEQUEST and proteome discoverer software. RESULTS: TGFß1 was identified in otosclerosis but not in the normal control temporal bone specimen. Aside from TGFß1, many proteins and predicted cDNA-encoded proteins were observed, with implications in cell death and/or proliferation pathways, suggesting a possible role in otosclerotic bone remodeling. Immunostaining using TGFß1 monoclonal revealed marked staining of the spongiotic otosclerotic lesions. CONCLUSIONS: Mechanisms involved in cochlear extension of otosclerosis are still unclear, but the implication of TGFß1 is supported by the present proteomic data and immunostaining results. The established role of TGFß1 in the chondrogenesis process supports the theory of a reaction targeting the globulae interossei within the otic capsule.

Neurofibromatosis 2 invasion of the internal auditory canal wall: clinical significance.

OBJECTIVE: To describe the infiltration of severe phenotype ("Wishart") neurofibromatosis type 2 (NF2)-related vestibular nerve schwannomas (VSs) into the internal auditory canal wall in contrast to sporadic VS and the milder ("Gardner") phenotype NF2-related VS. STUDY DESIGN: Retrospective case series involving microscopic examination and review of clinical history. SETTING: Temporal bone laboratory, harboring 849 documented pairs of decalcified, formalin-fixed, celloidin-embedded, sectioned human temporal bones (hTBs) with clinical history. SUBJECTS AND METHODS: Histologic sections from 56 patients who had been treated by the House Clinic for VS and who had pledged their temporal bones were identified in the data base of the laboratory. Twenty-four hTBs were from individuals with NF2.Each series of sections was examined microscopically for evidence of invasion of the walls of the internal auditory canal (IAC), hearing thresholds, speech discrimination, score (SDS), and tumor recurrence. RESULTS: Infiltration of the walls of the IAC by small buds of VS was found in 17 of the 24 NF2 hTBs. The only 2 NF2 without invasion were from an elderly patient with the milder (Gardner) form of NF2. Ten of the 12 NF2 patients had undergone surgery for the removal of their tumor, but residual tumor remained in the bone surrounding the IAC. Invasive VS were associated with poorer hearing thresholds at 250, 500, 1,000, and 2,000 Hz and lower SDS score. A relationship between invasion and recurrence was not statistically significant. CONCLUSION: The majority of IAC tumors associated with the severe "Wishart" phenotype demonstrate bone invasion within the IAC. Invasion of bone was associated with poorer hearing. The invasive nature of NF2-associated tumors may partially explain their higher recurrence rate after resection. Surgeons managing NF2-related VS should be aware of the small infiltrations of the wall of the IAC when removing these tumors to minimize recurrence.

The periductal channels of the endolymphatic duct, hydrodynamic implications.

OBJECTIVE: To describe the anatomy of a small network of channels surrounding the human endolymphatic duct. STUDY DESIGN: Archival temporal bone sections and a surgical specimen were studied using a variety of techniques. SETTING: Temporal bone laboratory of the House Research Institute. SUBJECTS AND METHODS: Archival temporal bone sections were examined by light microscopy, 3D reconstruction, and immunohistochemical labeling. A surgical specimen was examined using electron microscopy. Sections from temporal bones with blocked endolymphatic ducts or amputated sacs were examined for the manifestations of endolymphatic hydrops. RESULTS: Peri-endolymphatic duct channels were found to extend from the proximal cisternal part of the endolymphatic sac to the supporting tissue of the saccule and utricle. Tissue in the channels, as seen by conventional and electron microscopy, is continuous with and identical with the tissue surrounding the endolymphatic duct. Tissue in the channels labels with the S100 antibody similar to the spiral ligament and supporting tissue of the vestibular end organs and suggests a neural crest origin, as did the presence of melanocytes. Obstruction of the endolymphatic duct resulted in endolymphatic hydrops whereas amputation of the sac did not. CONCLUSION: Endolymph is probably absorbed in the endolymphatic duct. The peri-endolymphatic duct channels that extend from the proximal sac to the supporting tissue of the saccule label with the S100 antibody and contain melanocytes suggest a neural crest origin and involvement in fluid and potassium hydrodynamics similar to those described for the similarly staining spiral ligament of the cochlea.

Possible role of gap junction intercellular channels and connexin 43 in satellite glial cells (SGCs) for preservation of human spiral ganglion neurons : A comparative study with clinical implications.

Human spiral ganglion (SG) neurons show remarkable survival properties and maintain electric excitability for a long time after complete deafness and even separation from the organ of Corti, features essential for cochlear implantation. Here, we analyze and compare the localization and distribution of gap junction (GJ) intercellular channels and connexin 43 (Cx43) in cells surrounding SG cell bodies in man and guinea pig by using transmission electron microscopy and confocal immunohistochemistry. GJs and Cx43 expression has been recognized in satellite glial cells (SGCs) in non-myelinating sensory ganglia including the human SG. In man, SG neurons can survive as mono-polar or "amputated" cells with unbroken central projections following dendrite degeneration and consolidation of the dendrite pole. Cx43-mediated GJ signaling between SGCs is believed to play a key role in this "healing" process and could explain the unique preservation of human SG neurons and the persistence of cochlear implant function.