Planar cell polarity governs the alignment of the nasopharyngeal epithelium in mammals.

Planar cell polarity (PCP) signalling specifies the orientation of epithelial cells and regulates directional beating of motile cilia of multiciliated epithelial cells. Clinically, defects in cilia function are associated with nasopharyngeal symptoms. The polarity of the nasopharyngeal epithelium is poorly understood. Here, we demonstrated PCP in the nasopharyngeal epithelium. Multiciliated cells (MCCs) were uniformly aligned with their long axis parallel to the tissue axis of the nasopharynx (NP). In addition, PCP proteins exhibited an asymmetrical localisation between adjacent cells. Motile cilia were uniformly aligned in the same direction within both individual cells and neighbouring cells, which manifested as cilial polarity in MCCs. Mutation of Vangl2, a mammalian homologue of the Drosophila PCP gene, resulted in significant disruption of the orientation of epithelial cells. Finally, keratin-5-positive basal cells constantly replenished the luminal ciliated cells; the new dynamic ciliated cells were also oriented parallel to the tissue axis. These results indicate a role for the PCP pathway in the uniform orientation of dynamically replenished epithelial cells in the NP.

Concurrent Occurrence of Congenital Ossicular Anomaly and Localized Cholesteatoma: Series of 10 Cases.

OBJECTIVE: The objective of this study is to describe the clinical features, managements and outcomes of a rare coexistence of congenital ossicular anomaly and localized cholesteatoma. A literature review on these cases and each congenital disorder is also presented. METHODS: A retrospective chart review was performed on patients diagnosed with congenital ossicular anomaly with concurrent localized cholesteatoma from 2008 to 2017. Clinical data of these patients were collected. RESULTS: A total of 10 patients were identified. All patients presented with unilateral hearing loss. Pure-tone audiometry showed conductive hearing loss in all affected ears with an average air conduction (AC) threshold of 59 dB. High-resolution computed tomography scans of the temporal bone diagnosed ossicular anomaly for 90% (9/10); however, only 50% (5/10) had a diagnosis of localized cholesteatoma. A transcanal exploratory tympanotomy under the microscope was performed to discover whether the localized tiny-sized cholesteatoma around the ossicular chain did not have direct contact with the ossicular chain, which could be diagnosed as congenital cholesteatoma. We removed the localized cholesteatoma and reconstructed the ossicular chain in each patient. All localized cholesteatomas were found in the posterior-superior quadrant of the middle ear. Ossicular chain anomalies were associated with the incus and/or the stapes in all cases. Hearing improvement was achieved in each of the 6 patients who were followed up postoperatively, with an average AC threshold of 35 dB. The clinical features of congenital ossicular anomaly with concurrent congenital cholesteatoma were compared with those of each congenital disorder. The pathogenesis of each condition was also discussed. CONCLUSIONS: Congenital ossicular anomaly with concurrent congenital cholesteatoma is rare. It shares similar clinical features with congenital ossicular anomaly occurring alone, therefore awareness should be raised for a possible concurrent congenital cholesteatoma which was easy to miss in the diagnosis (50%) by the radiologist. A patient's hearing level can be improved by removal of the cholesteatoma and reconstruction of the ossicular chain. Localized cholesteatoma does not usually show residuals or recurrence.

Deep Learning in Automated Region Proposal and Diagnosis of Chronic Otitis Media Based on Computed Tomography.

OBJECTIVES: The purpose of this study was to develop a deep-learning framework for the diagnosis of chronic otitis media (COM) based on temporal bone computed tomography (CT) scans. DESIGN: A total of 562 COM patients with 672 temporal bone CT scans of both ears were included. The final dataset consisted of 1147 ears, and each of them was assigned with a ground truth label from one of the 3 conditions: normal, chronic suppurative otitis media, and cholesteatoma. A random selection of 85% dataset (n = 975) was used for training and validation. The framework contained two deep-learning networks with distinct functions: a region proposal network for extracting regions of interest from 2-dimensional CT slices; and a classification network for diagnosis of COM based on the extracted regions. The performance of this framework was evaluated on the remaining 15% dataset (n = 172) and compared with that of 6 clinical experts who read the same CT images only. The panel included 2 otologists, 3 otolaryngologists, and 1 radiologist. RESULTS: The area under the receiver operating characteristic curve of the artificial intelligence model in classifying COM versus normal was 0.92, with sensitivity (83.3%) and specificity (91.4%) exceeding the averages of clinical experts (81.1% and 88.8%, respectively). In a 3-class classification task, this network had higher overall accuracy (76.7% versus 73.8%), higher recall rates in identifying chronic suppurative otitis media (75% versus 70%) and cholesteatoma (76% versus 53%) cases, and superior consistency in duplicated cases (100% versus 81%) compared with clinical experts. CONCLUSIONS: This article presented a deep-learning framework that automatically extracted the region of interest from two-dimensional temporal bone CT slices and made diagnosis of COM. The performance of this model was comparable and, in some cases, superior to that of clinical experts. These results implied a promising prospect for clinical application of artificial intelligence in the diagnosis of COM based on CT images.

Mechanisms of Hearing Loss in a Guinea Pig Model of Superior Semicircular Canal Dehiscence.

Defective acoustic transmission in the cochlea is closely related with various auditory and vestibular symptoms. Among them, semicircular canal dehiscence (SCD) with a defective semicircular bone is typical. Currently, the pathogenesis of SCD is usually explained by the third window hypothesis; however, this hypothesis fails to explain the variability in the symptoms and signs experienced by superior SCD (SSCD) patients. We evaluated the mechanism of hearing loss in a guinea pig model of bony dehiscence with various sizes and locations along the superior semicircular canal. Auditory brainstem responses (ABRs) and laser Doppler velocimetry were used to measure hearing loss and vibration changes before and after fenestration, as well as after restorative patching. ABR thresholds at low frequencies (e.g., 1000 Hz) increased after fenestration and decreased back to the normal range after we repaired the defect. Energy leakage from the surgically introduced third window was detected in the range of 300-1500 Hz, accompanied by increased vibration at the umbo, stapes head, and the dehiscence site, while decreased vibration was observed at the round window membrane in the same frequency range. After the patching procedure, the deviant vibrations were recovered. The degree of postfenestration energy leakage was proportional to the size of fenestration and the proximity of the fenestration site to the oval window. These results suggest that the bony fenestration of the superior semicircular canal mimics the hearing loss pattern of patients with SSCD. The decrease in perilymph wave impedance likely accounts for the auditory changes.

Cell proliferation during hair cell regeneration induced by Math1 in vestibular epithelia in vitro.

Hair cell regeneration is the fundamental method of correcting hearing loss and balance disorders caused by hair cell damage or loss. How to promote hair cell regeneration is a hot focus in current research. In mammals, cochlear hair cells cannot be regenerated and few vestibular hair cells can be renewed through spontaneous regeneration. However, Math1 gene transfer allows a few inner ear cells to be transformed into hair cells in vitro or in vivo. Hair cells can be renewed through two possible means in birds: supporting cell differentiation and transdifferentiation with or without cell division. Hair cell regeneration is strongly associated with cell proliferation. Therefore, this study explored the relationship between Math1-induced vestibular hair cell regeneration and cell division in mammals. The mouse vestibule was isolated to harvest vestibular epithelial cells. Ad-Math1-enhanced green fluorescent protein (EGFP) was used to track cell division during hair cell transformation. 5-Bromo-2'-deoxyuridine (BrdU) was added to track cell proliferation at various time points. Immunocytochemistry was utilized to determine cell differentiation and proliferation. Results demonstrated that when epithelial cells were in a higher proliferative stage, more of these cells differentiated into hair cells by Math1 gene transfer. However, in the low proliferation stage, no BrdU-positive cells were seen after Math1 gene transfer. Cell division always occurred before Math1 transfection but not during or after Math1 transfection, when cells were labeled with BrdU before and after Ad-Math1-EGFP transfection. These results confirm that vestibular epithelial cells with high proliferative potential can differentiate into new hair cells by Math1 gene transfer, but this process is independent of cell proliferation.

Junctional E-cadherin/p120-catenin Is Correlated with the Absence of Supporting Cells to Hair Cells Conversion in Postnatal Mice Cochleae.

Notch inhibition is known to generate supernumerary hair cells (HCs) at the expense of supporting cells (SCs) in the mammalian inner ear. However, inhibition of Notch activity becomes progressively less effective at inducing SC-to-HC conversion in the postnatal cochlea and balance organs as the animal ages. It has been suggested that the SC-to-HC conversion capacity is inversely correlated with E-cadherin accumulation in postnatal mammalian utricles. However, whether E-cadherin localization is linked to the SC-to-HC conversion capacity in the mammalian inner ear is poorly understood. In the present study, we treated cochleae from postnatal day 0 (P0) with the Notch signaling inhibitor DAPT and observed apparent SC-to-HC conversion along with E-cadherin/p120ctn disruption in the sensory region. In addition, the SC-to-HC conversion capacity and E-cadherin/p120ctn disorganization were robust in the apex but decreased toward the base. We further demonstrated that the ability to regenerate HCs and the disruption of E-cadherin/p120ctn concomitantly decreased with age and ceased at P7, even after extended DAPT treatments. This timing is consistent with E-cadherin/p120ctn accumulation in the postnatal cochleae. These results suggest that the decreasing capacity of SCs to transdifferentiate into HCs correlates with E-cadherin/p120ctn localization in the postnatal cochleae, which might account for the absence of SC-to-HC conversion in the mammalian cochlea.

Afferent synaptogenesis between ectopic hair-cell-like cells and neurites of spiral ganglion induced by Atoh1 in mammals in vitro.

Newly formed ectopic hair-cell-like cells (EHCLCs) induced by overexpression of atonal homolog 1 (Atoh1) in vitro were found to possess features of endogenous hair cells (HCs) in previous reports and in the present study. However, limited information is available regarding whether EHCLCs and native spiral ganglion neurons (SGNs) form afferent synapses, which are important for the restoration of hearing. In the current study, we focused on the afferent synaptogenesis between EHCLCs and SGN-derived dendrites. Cochlear explants of auditory epithelia with native SGNs retained were cultured in vitro, and human adenovirus serotype 5 (Ad5) vectors encoding Atoh1 were used to overexpress Atoh1 and induce EHCLCs. We observed that the neurites of the original SGNs extended toward the lesser epithelial ridge (LER) and innervated the EHCLCs. Immunohistochemical analyses revealed the expression of presynaptic ribbon C-terminal-binding protein 2 (CtBP2) and postsynaptic density protein (PSD)-95 in the nerve endings of SGN-derived neurons adjacent to EHCLCs. PSD-95 was located directly opposite CtBP2-positive puncta in the terminals of branches of SGNs, demonstrating that the neurites of SGNs formed afferent-like synaptic connections with EHCLCs. However, the expression of glutamate receptor type 2 (GluR2) could not be detected in the terminals of branches of SGNs surrounding EHCLCs. In addition, we found that the presynaptic ribbon (CtBP2) formation in EHCLCs preceded neural innervation. Furthermore, CtBP2-positive puncta increased and then decreased in EHCLCs, similar to the changes observed in endogenous HCs in terms of their number and distribution. Our finding of the generation of cochlear afferent synapses between EHCLCs and original SGNs will lay the foundation for regenerative approaches to restoring hearing after hair cell loss.

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae.

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Notch signaling is active in normal mouse middle ear epithelial cells.

Mucous cell metaplasia/hyperplasia in the middle ear epithelium is associated with the occurrence of otitis media with effusion during infections. However, the mechanism by which Notch signaling regulates cell fate in the middle ear epithelium is unclear. The aim of the present study was to elucidate this mechanism by investigating the localization of Notch receptors, such as Notch1 and Notch2, and Notch ligands, such as Jagged1, in the normal mouse middle ear epithelium (NMMEE) using immunofluorescence. Furthermore, the mRNA expression levels of Notch receptors and ligands were evaluated using reverse transcription polymerase chain reaction (PCR). The effects of the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT) on epithelial cell proliferation were determined using 5-ethynyl-2'-deoxyuridine (EdU) staining and immunofluorescence staining of the apoptosis marker caspase-3 and the epithelial proliferation marker pan-cytokeratine. In addition, the differentiation of the NMMEE cells was characterized by evaluating the mRNA expression levels of the mucous cell-associated genes Arg2, Muc2, Spdef, Spink4 and Tff1 using quantitative PCR. Notch1, Notch2 and Jagged1 were observed to be co-localized throughout the mouse middle ear epithelium. Furthermore, Notch1-4, Jagged1, Jagged2, Dll1 and Dll4 mRNAs were expressed in the NMMEE cells. The inhibition of Notch by DAPT resulted in fewer EdU-positive cells and the upregulation of the expression levels of various mucous cell-associated genes. The results indicate that DAPT suppresses the proliferation of NMMEE cells while promoting their differentiation into mucous cells. Therefore, DAPT may provide a specific therapeutic strategy for the reversal of multiple pathological processes that are associated with epithelium thickening in the middle ear.

Shrinkage of ipsilateral taste buds and hyperplasia of contralateral taste buds following chorda tympani nerve transection.

The morphological changes that occur in the taste buds after denervation are not well understood in rats, especially in the contralateral tongue epithelium. In this study, we investigated the time course of morphological changes in the taste buds following unilateral nerve transection. The role of the trigeminal component of the lingual nerve in maintaining the structural integrity of the taste buds was also examined. Twenty-four Sprague-Dawley rats were randomly divided into three groups: control, unilateral chorda tympani nerve transection and unilateral chorda tympani nerve transection + lingual nerve transection. Rats were allowed up to 42 days of recovery before being euthanized. The taste buds were visualized using a cytokeratin 8 antibody. Taste bud counts, volumes and taste receptor cell numbers were quantified and compared among groups. No significant difference was detected between the chorda tympani nerve transection and chorda tympani nerve transection + lingual nerve transection groups. Taste bud counts, volumes and taste receptor cell numbers on the ipsilateral side all decreased significantly compared with control. On the contralateral side, the number of taste buds remained unchanged over time, but they were larger, and taste receptor cells were more numerous postoperatively. There was no evidence for a role of the trigeminal branch of the lingual nerve in maintaining the structural integrity of the anterior taste buds.

Role of the planar cell polarity pathway in regulating ectopic hair cell-like cells induced by Math1 and testosterone treatment.

Planar cell polarity (PCP) signaling regulates cochlear extension and coordinates orientation of sensory hair cells in the inner ear. Retroviral-mediated introduction of the Math1 transcription factor leads to the transdifferentiation of some mature supporting cells into hair cells. Testosterone, a gonadal sex steroid hormone, is associated with neuroprotection and regeneration in Central Nervous System (CNS) development. Experiments were performed in vitro using Ad5-EGFP-Math1/Ad5-Math1 in neonatal mouse cochleas. Establishment of ectopic hair-cell like cell(HCLC) polarity in the lesser epithelial ridge (LER) with or without testosterone-3-(O-carboxymethyl) oxime bovine serum albumin (testosterone-BSA) treatment was investigated to determine the role of the PCP pathway in regulating ectopic regenerated (HCLCs) through induction by Math1 and testosterone treatment. After Math1 infection, new ectopic regenerated HCLCs were detected in the LER. After the HCLCs developed actin-rich stereocilia, the basal bodies moved from the center to the distal side. Moreover, the narrower, non-sensory LER region meant that the convergent extension (CE) was also established after transfection with Math1. After 9 days of in vitro testosterone-BSA treatment, more Edu(+), Sox2(+), and HCLC cells were observed in the LER with an accompanying downregulation of E-cadherin. Interestingly, the CE of the Ad5-EGFP-math1 treated LER is altered, but the intrinsic cellular polarity of the HCLCs is not obviously changed. In summary, our results indicate that PCP signaling is involved in the development of ectopic HCLCs and the CE of the ectopic sensory region is altered by testosterone-BSA through downregulation of cell-cell adhesion. Testosterone-BSA and Math1 treatment could promote an increase in HCLCs in the LER through proliferation and transdifferentiation.

Atoh1 expression levels define the fate of rat cochlear nonsensory epithelial cells in vitro.

Atonal homolog 1 (Atoh1) is a basic helix­loop­helix transcription factor that is essential for inner ear hair cell differentiation. Previous studies have reported that Atoh1 gene transfer induces the production of ectopic hair cell­like cells (EHCLCs). In the present study, the effect of different Atoh1 expression levels and the duration of EHCLC formation on the lesser epithelial ridge (LER) of cochleae was examined using a human adenovirus serotype 5 (Ad5) vector encoding atoh1 and the reporter gene EGFP. Different Ad5­EGFP­atoh1/Ad5­EGFP virus titers were added to cultured cochlear explants and EHCLCs were detected in the LER at various time points. The results demonstrated that GFP alone did not induce EHCLCs. By contrast, Atoh1 expression induced EHCLCs as early as 2.5­5 days following EGFP­atoh1 infection in the LER and depending upon the viral titer, the number of EHCLCs increased with time. Higher Ad5­EGFP­atoh1 titers induced enhanced Atoh1 expression, resulting in an increase in EHCLCs. Lower Ad5­EGFP­atoh1 titers required more time for EHCLC formation and very low titers of Ad5­EGFP­atoh1 induced only weak Atoh1 expression and did not trigger EHCLC formation. In conclusion, the present study utilized an appropriate Ad5­EGFP­atoh1 titer range to induce Atoh1 expression and the subsequent production of EHCLCs. The results revealed that the Atoh1 expression level defined the fate of LER cells as either EHCLCs or nonsensory epithelial cells. This evidence may provide an important guideline for future studies into gene therapy strategies for the treatment of deafness.

Changes in ADF/destrin expression in the development of hair cells following Atoh1-induced ectopic regeneration.

The aim of this study was to investigate the effects of actin depolymerizing factor (ADF)/destrin and position changes of kinetosomes in the development of hair cells following Atoh1-induced ectopic regeneration in the basilar membrane of mice. We observed through immunofluorescence at various time-points the expression of ADF/destrin and the specific kinetosome marker, γ-tubulin, in hair cells following ectopic regeneration induced by adenovirus transfection, overexpression of Atoh1 and in vitro culture. Changes of ADF/destrin distribution and kinetosome position during in vitro culture of new hair cells [Myo7a(+)] following Atoh1-induced ectopic regeneration are consistent with the changes in ADF/destrin expression and the polar migration of kinetosomes in hair cells of the cochlear sensory epithelium in normal development. ADF/destrin is involved in the development of the auditory epithelium and the development and structural rearrangement of ectopically regenerated hair cells in mammals. The kinetosomes of hair cells following Atoh1-induced ectopic regeneration show positional changes in vitro at different time-points.

Clinical characteristics of patients with labyrinthine fistulae caused by middle ear cholesteatoma.

BACKGROUND: Labyrinthine fistula (LF) is a very common clinical complication mainly caused by middle ear cholesteatoma. Whether the presence of different degree LF caused by middle ear cholesteatoma aggravates neurosensory hearing loss (NSHL) and what is the degree of the hearing loss caused by LF were still under controversial. This study aimed to investigate whether the LF degree is correlative with the age distribution, disease duration and hearing loss degree for cholesteatomatous patients. METHODS: The files of 143 patients with middle ear cholesteatoma were selected and reviewed in a retrospective study. Seventy-eight patients with LF were divided into three types according to the degree of destruction of labyrinth. Sixty-five patients without LF were randomly chosen for control. Then, we compared the clinical characteristics of patients with or without labyrinthine fistulae caused by middle ear cholesteatoma. RESULTS: According to the study, cholesteatomatous patients with LF were older and suffered longer disease duration than those without LF. Hearing loss is severe with high frequencies both in patients with and without LF. Moreover, inner ear impairment is correlative with the degree of destruction in labyrinth, and more severe destruction in labyrinth follow the more severe symptoms correlative with inner ear impairment. CONCLUSION: Surgical intervention should be performed as early as possible for these cholesteatomatous patients.

Cutaneous neuroendocrine carcinoma of the external auditory canal: a case report and review of the literature.

Cutaneous neuroendocrine carcinoma (cNEC) is rarely seen in the external ear. In this paper, we newly describe a patient with cNEC in his right external auditory canal, followed by a further discussion on the clinical features, diagnosis, and treatments of cNEC of the external ear. A review of the literature showed that cNEC of the external auditory canal generally presents as asymptomatic and that pathology yields the most confirmative diagnosis. A wide resection with adjuvant radiotherapy and chemotherapy is recommended. The overall prognosis of this condition is poor.

Expression of Numb and Numb-like in the development of mammalian auditory sensory epithelium.

The Numb and Numb-like are evolutionarily conserved cell fate-determining factors in mammals. For the first time, we investigate the involvement of the Numb and Numb-like in the developing auditory sensory epithelium. We show that both of them are expressed in the rat auditory sensory epithelium, and the four isoforms of the Numb have dynamic expression patterns during cochlear development. At the early stage of the auditory epithelium development, they occur in all progenitor cells. At the late stage of cell differentiation, they are expressed mainly in the cytoplasm of apical cells and their locations are different. Furthermore, we find overexpression of the Numb or Numb-like, in cochlear whole mount cultures, can upregulate mRNA level of Rath1, which is important in the hair-cell development.

Survival and fate of transplanted embryonic neural stem cells by Atoh1 gene transfer in guinea pigs cochlea.

Embryonic neural stem cells (NSCs) were isolated from the neuroepithelium of the dorsal telencephalon of embryonic rats and infected by Ad5-Atoh1-enhanced green fluorescent protein. These NSCs were then delivered into neurosphere culture medium or transplanted into the endolymphatic space of the normal guinea pig cochlea through cochleostomy. Embryonic NSC phenotype of these isolated cells was determined by immunohistochemical detection of cell-specific protein markers. Survival, location and hair cell (HC) differentiation of the implanted NSCs were determined by the expression of the report gene, enhanced green fluorescent protein, and a specific marker for HCs, Myosin VIIa. These implanted cells can survive in the endolymphatic space of the cochlea. Some of the surviving cells differentiated into HCs by Atoh1 gene transfer.