Lineage-tracing and translatomic analysis of damage-inducible mitotic cochlear progenitors identifies candidate genes regulating regeneration.

Cochlear supporting cells (SCs) are glia-like cells critical for hearing function. In the neonatal cochlea, the greater epithelial ridge (GER) is a mitotically quiescent and transient organ, which has been shown to nonmitotically regenerate SCs. Here, we ablated Lgr5+ SCs using Lgr5-DTR mice and found mitotic regeneration of SCs by GER cells in vivo. With lineage tracing, we show that the GER houses progenitor cells that robustly divide and migrate into the organ of Corti to replenish ablated SCs. Regenerated SCs display coordinated calcium transients, markers of the SC subtype inner phalangeal cells, and survive in the mature cochlea. Via RiboTag, RNA-sequencing, and gene clustering algorithms, we reveal 11 distinct gene clusters comprising markers of the quiescent and damaged GER, and damage-responsive genes driving cell migration and mitotic regeneration. Together, our study characterizes GER cells as mitotic progenitors with regenerative potential and unveils their quiescent and damaged translatomes.

The impact of targeted ablation of one row of outer hair cells and Deiters' cells on cochlear amplification.

The mammalian cochlea contains three rows of outer hair cells (OHCs) that amplify the basilar membrane traveling wave with high gain and exquisite tuning. The pattern of OHC loss caused by typical methods of producing hearing loss in animal models (noise, ototoxic exposure, or aging) is variable and not consistent along the length of the cochlea. Thus, it is difficult to use these approaches to understand how forces from multiple OHCs summate to create normal cochlear amplification. Here, we selectively removed the third row of OHCs and Deiters' cells in adult mice and measured cochlear amplification. In the mature cochlear epithelia, expression of the Wnt target gene Lgr5 is restricted to the third row of Deiters' cells, the supporting cells directly underneath the OHCs. Diphtheria toxin administration to Lgr5DTR-EGFP/+ mice selectively ablated the third row of Deiters' cells and the third row of OHCs. Basilar membrane vibration in vivo demonstrated disproportionately lower reduction in cochlear amplification by about 13.5 dB. On a linear scale, this means that the 33% reduction in OHC number led to a 79% reduction in gain. Thus, these experimental data describe the impact of reducing the force of cochlear amplification by a specific amount. Furthermore, these data argue that because OHC forces progressively and sequentially amplify the traveling wave as it travels to its peak, the loss of even a relatively small number of OHCs, when evenly distributed longitudinally, will cause a substantial reduction in cochlear amplification.NEW & NOTEWORTHY Normal cochlear physiology involves force production from three rows of outer hair cells to amplify and tune the traveling wave. Here, we used a genetic approach to target and ablate the third row of outer hair cells in the mouse cochlea and found it reduced cochlear amplification by 79%. This means that the loss of even a relatively small number of OHCs, when evenly distributed, causes a substantial reduction in cochlear amplification.

Retrograde mastoidectomy with canal wall reconstruction versus intact canal wall tympanomastoidectomy for cholesteatoma with minimal mastoid extension.

PURPOSE: Appropriate reconstruction of the canal wall or maintenance of the middle ear pressure in cholesteatoma may help in preventing recurrence. Retrograde mastoidectomy with canal wall reconstruction (RMR) can overcome the challenge of a wider canal wall defect or temporal bone immaturity, which possibly increases the recurrence risk. This study compared the outcomes of RMR and intact canal wall tympanomastoidectomy (ICW) for cholesteatomas with minimal mastoid extension and quantitatively evaluate the relationship between anatomical features and recurrence. METHODS: This single-center retrospective cohort study included patients who had undergone primary ICW or RMR for pars flaccida cholesteatoma with minimal mastoid extension from 2009 to 2019. The main outcome measures were anatomical measurements of the shortest distance between the cranial fossa and the upper canal wall (SCU), attic volume, and bony defect area of the canal wall (BDC) on computed tomography; recidivism; and postoperative air-bone gap (ABG). RESULTS: There were no significant differences in the preoperative anatomical factors, recidivism incidence, and postoperative ABG between the RMR (n = 20) and ICW (n = 60) groups. However, the median BDC was significantly greater in the RMR group (58.3 vs. 37.0 mm2). There was no significant difference in the SCU and attic volume between patients with and without recurrence. CONCLUSION: Selection of RMR or ICW may not affect recidivism and hearing outcomes in cholesteatoma with minimal mastoid extension. Bony defect size and attic narrowness were not associated with recurrence. Considering wider visualization and one-staged operation, RMR can be more adaptable than ICW for cholesteatoma with minimal mastoid extension.

Loss of Pax3 causes reduction of melanocytes in the developing mouse cochlea.

Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of skin and retinal melanocytes, manifested as congenital hearing loss (~ 70%) and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. Cochlear melanocytes in the stria vascularis originated from Pax3-traced melanoblasts and Plp1-traced Schwann cell precursors, both of which derive from neural crest cells. Here, using a Pax3-Cre knock-in mouse that allows lineage tracing of Pax3-expressing cells and disruption of Pax3, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3+ derivatives contribute to S100+, Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all of which are significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in humans.

Pax3 deficiency diminishes melanocytes in the developing mouse cochlea.

Cochlear melanocytes are intermediate cells in the stria vascularis that generate endocochlear potentials required for auditory function. Human PAX3 mutations cause Waardenburg syndrome and abnormalities of melanocytes, manifested as congenital hearing loss and hypopigmentation of skin, hair and eyes. However, the underlying mechanism of hearing loss remains unclear. During development, cochlear melanocytes in the stria vascularis are dually derived from Pax3-Cre+ melanoblasts migrating from neuroepithelial cells including neural crest cells and Plp1+ Schwann cell precursors originated from also neural crest cells, differentiating in a basal-apical manner. Here, using a Pax3-Cre mouse line, we found that Pax3 deficiency causes foreshortened cochlea, malformed vestibular apparatus, and neural tube defects. Lineage tracing and in situ hybridization show that Pax3-Cre derivatives contribute to S100+ , Kir4.1+ and Dct+ melanocytes (intermediate cells) in the developing stria vascularis, all significantly diminished in Pax3 mutant animals. Taken together, these results suggest that Pax3 is required for the development of neural crest cell-derived cochlear melanocytes, whose absence may contribute to congenital hearing loss of Waardenburg syndrome in human.

Imaging alloreactive T cells provides early warning of organ transplant rejection.

Diagnosis of organ transplant rejection relies upon biopsy approaches to confirm alloreactive T cell infiltration in the graft. Immune molecular monitoring is under investigation to screen for rejection, though these techniques have suffered from low specificity and lack of spatial information. ImmunoPET utilizing antibodies conjugated to radioisotopes has the potential to improve early and accurate detection of graft rejection. ImmunoPET is capable of noninvasively visualizing the dynamic distribution of cells expressing specific immune markers in the entire body over time. In this work, we identify and characterize OX40 as a surrogate biomarker for alloreactive T cells in organ transplant rejection and monitor its expression by utilizing immunoPET. In a dual murine heart transplant model that has both syngeneic and allogeneic hearts engrafted in bilateral ear pinna on the recipients, OX40 immunoPET clearly depicted alloreactive T cells in the allograft and draining lymph node that were not observed in their respective isograft counterparts. OX40 immunoPET signals also reflected the subject's immunosuppression level with tacrolimus in this study. OX40 immunoPET is a promising approach that may bridge molecular monitoring and morphological assessment for improved transplant rejection diagnosis.

Sox2 haploinsufficiency primes regeneration and Wnt responsiveness in the mouse cochlea.

During development, Sox2 is indispensable for cell division and differentiation, yet its roles in regenerating tissues are less clear. Here, we used combinations of transgenic mouse models to reveal that Sox2 haploinsufficiency (Sox2haplo) increases rather than impairs cochlear regeneration in vivo. Sox2haplo cochleae had delayed terminal mitosis and ectopic sensory cells, yet normal auditory function. Sox2haplo amplified and expanded domains of damage-induced Atoh1+ transitional cell formation in neonatal cochlea. Wnt activation via ß-catenin stabilization (ß-cateninGOF) alone failed to induce proliferation or transitional cell formation. By contrast, ß-cateninGOF caused proliferation when either Sox2haplo or damage was present, and transitional cell formation when both were present in neonatal, but not mature, cochlea. Mechanistically, Sox2haplo or damaged neonatal cochleae showed lower levels of Sox2 and Hes5, but not of Wnt target genes. Together, our study unveils an interplay between Sox2 and damage in directing tissue regeneration and Wnt responsiveness and thus provides a foundation for potential combinatorial therapies aimed at stimulating mammalian cochlear regeneration to reverse hearing loss in humans.

[Effects of ESS on sinobronchial syndrome].

Sinobronchial syndrome (SBS) is characterized by chronic sinusitis with chronic infection of the bronchus. Chronic bronchitis, bronchiectasis and diffuse panbronchiolitis are also examples of complicating lower airway disease. In Japan, the surgical treatment of sinusitis is not considered to improve the lower airway diseases. Most published reports describe the Caldwell-Luc method. However ESS (Endoscopic Sinus Surgery) can heal sinusitis while maintaining the morphology and function of the paranasal sinus. In addition, its surgical results are satisfactory. This report describes the improvement of patients with SBS who underwent ESS. Twelve patients with sinobronchial syndrome who underwent ESS at our department between 1989 and 1993 were enrolled in the study. Subjective improvement in sinusitis and the lower airway diseases were evaluated using a questionnaire. Objective improvements in sinusitis were evaluated using endoscopic findings of the ethmoid sinuses, while objective improvements in the lower airway diseases were evaluated by measuring the vital capacity and FEV 1.0%. These examinations were performed one year after the operation; the results of long-term follow-up examinations performed more than 6 years after the operation were also studied. In all of the patients, subjective nasal symptoms and objective endoscopic findings were satisfactory, with improvement rates of 91.7% and 83.3%, respectively. Regarding the lower airway symptoms, all the patients subjectively rated the symptoms as having improved, and an improvement in the FEV 1.0% was improved in all of the cases. In the present study, the lower airway symptoms improved after ESS. ESS enables the paranasal sinuses to be treated while maintaining the morphology of the paranasal sinus. Consequently, surgical results are better. The improvement of chronic sinusitis reduces the direct inflow of postnasal drippings into the bronchus. Restoring the normal functions of the nasal sinus defends the lower airway by warning, and humidifying the inspiratory air and removing dusts; these functions are though to have a favorable effect on the lower airway.