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.

Histopathologic Characteristics of Internal Auditory Canal Diverticula.

HYPOTHESIS: We hypothesize that internal auditory canal (IAC) diverticula occur independent of otosclerosis as demonstrated by temporal bone histopathology. BACKGROUND: Diverticula at the anterior-inferior aspect of the IAC have been described histologically in the setting of cavitary otosclerosis. Recent radiographic studies show the prevalence of IAC diverticula that is higher than what can be accounted for by cavitary otosclerosis alone. METHODS: We examined hematoxylin and eosin temporal bone histopathology slides with otosclerosis involving the IAC. We also examined bones from normal hearing subjects with normal histologic findings. Temporal bones were included if donors were more than 18 years of age at time of death and adequate horizontal cuts were available to evaluate the area of interest. RESULTS: IAC diverticula were found in 33 of 47 (70%) temporal bones with IAC otosclerosis and in 5 of 20 (25%) normal temporal bones. The difference in mean pure tone averages (PTA) in the normal temporal bones with (PTA 7.3 ±â€Š7) and without (PTA 8 ±â€Š2) diverticula was not statistically significant (p = 0.86). CONCLUSION: IAC diverticula which have been previously demonstrated to occur in the setting of cavitary otosclerosis can also occur independent from otosclerosis. Subjects with diverticula but without other temporal bone pathology have normal hearing thresholds.

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.

Special Anatomic Considerations in Otosclerosis Surgery.

The anatomy of the vestibular organs together with considerations of the middle and inner ear anatomy relevant to stapes surgery is discussed. An archival collection of macerated and freshly frozen human temporal bones underwent micro-computed tomography (CT) with subsequent volume-rendering. Three-dimensional (3D) reconstructions and the topographic anatomy of the oval window were considered. Micro-CT and 3D rendering revealed the relationship between the otolith organs and the oval window. Anatomic variations were extensive and included the distance between the footplate and the reconstructed macula margins. A "no-go" zone is suggested for the surgeon to avoid injury during stapes surgery.

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.

Lack of Evidence for Nonotosclerotic Stapes Fixation in Human Temporal Bone Histopathology.

HYPOTHESIS: Nonotosclerotic stapes fixation does not represent a significant cause of stapes ankylosis in patients undergoing stapedectomy; the vast majority have otosclerosis. BACKGROUND: Nonotosclerotic stapes fixation has been proposed as the diagnosis in 30 to 40% of patients undergoing stapedectomy (after excluding rare congenital, systemic, and syndromic causes of stapes fixation and tympanosclerosis). This finding was based on the histopathologic evaluation of total stapedectomy surgical specimens. Since these specimens do not include the surrounding otic capsule, the histopathologic evidence of otosclerosis may be missed. METHODS: Human temporal bone specimens from patients who underwent stapes mobilization, stapedotomy, or stapedectomy during life were evaluated for histologic evidence of otosclerosis. Patients with a history of temporal bone trauma, tympanosclerosis, and congenital, systemic, or syndromic causes of stapes fixation were excluded. Therefore, most temporal bone donors carried a clinical diagnosis of otosclerosis. RESULTS: Two hundred ten specimens from three temporal bone collections were independently evaluated. Otosclerosis was found on histology in 99% (207/210). Therefore, the incidence of nonotosclerotic stapes fixation was 1% (3/210). In two of the three patients who did not have otosclerosis, the contralateral temporal bone had otosclerosis on histopathologic evaluation. These patients may have had otosclerosis in the footplate only (which was removed at the time of surgery and not available for review). CONCLUSION: Nonotosclerotic stapes fixation is not likely a distinct pathologic classification from otosclerosis. Most patients diagnosed with nonotosclerotic stapes fixation likely have otosclerosis, but do not have otosclerotic foci in the stapes itself.

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.