The lack of cochlear regenerative potential is the main cause for the permanence of hearing loss. Albeit quiescent in vivo, dissociated non-sensory cells from the neonatal cochlea proliferate and show ability to generate hair cell-like cells in vitro. Only a few non-sensory cell-derived colonies, however, give rise to hair cell-like cells, suggesting that sensory progenitor cells are a subpopulation of proliferating non-sensory cells. Here we purify from the neonatal mouse cochlea four different non-sensory cell populations by fluorescence-activated cell sorting (FACS). All four populations displayed proliferative potential, but only lesser epithelial ridge and supporting cells robustly gave rise to hair cell marker-positive cells. These results suggest that cochlear supporting cells and cells of the lesser epithelial ridge show robust potential to de-differentiate into prosensory cells that proliferate and undergo differentiation in similar fashion to native prosensory cells of the developing inner ear.

Otoferlin is essential for fast Ca2+-triggered transmitter release from auditory inner hair cells (IHCs), playing key roles in synaptic vesicle release, replenishment and retrieval. Dysfunction of otoferlin results in profound prelingual deafness. Despite its crucial role in cochlear synaptic processes, mechanisms regulating otoferlin activity have not been studied to date. Here, we identified Ca2+/calmodulin-dependent serine/threonine kinase II delta (CaMKIIδ) as an otoferlin binding partner by pull-downs from chicken utricles and reassured interaction by a co-immunoprecipitation with heterologously expressed proteins in HEK cells. We confirmed the expression of CaMKIIδ in rodent IHCs by immunohistochemistry and real-time PCR. A proximity ligation assay indicates close proximity of the two proteins in rat IHCs, suggesting that otoferlin and CaMKIIδ also interact in mammalian IHCs. In vitro phosphorylation of otoferlin by CaMKIIδ revealed ten phosphorylation sites, five of which are located within C2-domains. Exchange of serines/threonines at phosphorylated sites into phosphomimetic aspartates reduces the Ca2+ affinity of the recombinant C2F domain 10-fold, and increases the Ca2+ affinity of the C2C domain. Concordantly, we show that phosphorylation of otoferlin and/or its interaction partners are enhanced upon hair cell depolarization and blocked by pharmacological CaMKII inhibition. We therefore propose that otoferlin activity is regulated by CaMKIIδ in IHCs.

SummaryAlthough sevoflurane is commonly used in anaesthesia, a threshold value for maximum exposure to personnel does not exist and although anaesthetists are aware of the problem, surgeons rarely focus on it. We used a photo-acoustic infrared device to measure the exposure of surgeons to sevoflurane during paediatric adenoidectomies. Sixty children were randomly allocated to laryngeal mask, cuffed tracheal tube or uncuffed tracheal tube. The average mean (maximum) sevoflurane concentrations within the surgeons' operating area were 1.05 (10.05) ppm in the laryngeal mask group, 0.33 (1.44) ppm in the cuffed tracheal tube group and 1.79 (18.02) ppm in the uncuffed tracheal tube group, (p < 0.001), laryngeal mask and cuffed tracheal tube groups vs. uncuffed tube group. The presence of sevoflurane was noticed by surgeons in 20% of cases but there were no differences between the groups (p = 0.193). Surgical and anaesthetic complications were similar in all three groups. We conclude that sevoflurane can be safely used during adenoidectomies with all three airway devices, but in order to minimise sevoflurane peak concentrations, cuffed tracheal tubes are preferred.

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