Sensory hair cells located in the organ of Corti are essential for cochlear mechanosensation. Their loss is irreversible in humans resulting in permanent hearing loss. The development of therapeutic interventions for hearing loss requires fundamental knowledge about similarities and potential differences between animal models and human development as well as the establishment of human cell based-assays. Here we analyze gene and protein expression of the developing human inner ear in a temporal window spanning from week 8 to 12 post conception, when cochlear hair cells become specified. Utilizing surface markers for the cochlear prosensory domain, namely EPCAM and CD271, we purify postmitotic hair cell progenitors that, when placed in culture in three-dimensional organoids, regain proliferative potential and eventually differentiate to hair cell-like cells in vitro. These results provide a foundation for comparative studies with otic cells generated from human pluripotent stem cells and for establishing novel platforms for drug validation.

Hearing loss is an important sequela of pneumococcal meningitis (PM), occurring in up to 30% of survivors. The role of the severity of infection on hearing function and pathomorphological consequences in the cochlea secondary to PM have not been investigated to date. Using a well-established model of PM, we systematically investigated the functional hearing outcome and the long-term fate of neurosensory cells in the cochlea, i.e., hair cells and spiral ganglion neurons (SGNs), with a focus on their tonotopic distribution. Intracisternal infection of infant rats with increasing inocula of Streptococcus pneumoniae resulted in a dose-dependent increase in CSF levels of interleukin-1␤, interleukin-6, tumor necrosis factor ␣, interleukin-10, and interferon-␥ in acute disease. The severity of long-term hearing loss at 3 weeks after infection, measured by auditory brainstem response recordings, correlated to the initial inoculum dose and to the levels of proinflammatory cytokines determined in the acute phase of PM. Quantitative cochlear histomorphology revealed a significant loss of SGNs and outer hair cells that strongly correlated to the level of infection, with the most severe damage occurring in the basal part of the cochlea. Inner hair cells (IHCs) were not significantly affected throughout the entire cochlea. However, surviving IHCs lost synaptic connectivity to remaining SGNs in all cochlear regions. These findings provide evidence that the inoculum concentration, i.e., severity of infection, is the major determinant of long-term morphological cell pathologies in the cochlea and functional hearing loss.

BackgroundPneumococcal meningitis is associated with high mortality and morbidity rates. Up to 50% of survivors show neurologic sequelae including hearing loss, cognitive impairments and learning disabilities, being particularly detrimental in affected infants and children where adjuvant therapy with dexamethasone has no proven beneficial effect. We evaluated the effect of concomitantly targeting specific pathophysiological mechanisms responsible for brain damage—i.e. matrix-metalloproteinase (MMP) activity and the exacerbated cerebral inflammation provoked through antibiotic-induced bacterial lysis. Here, we combined adjunctive therapies previously shown to be neuroprotective when used as single adjuvant therapies.MethodsEleven-day-old Wistar rats were infected intracisternally with 6.44 ± 2.17 × 103 CFU Streptococcus pneumoniae and randomised for treatment with ceftriaxone combined with (a) single adjuvant therapy with daptomycin (n = 24), (b) single adjuvant therapy with Trocade (n = 24), (c) combined adjuvant therapy (n = 66) consisting of daptomycin and Trocade, or (d) ceftriaxone monotherapy (n = 42). Clinical parameters and inflammatory CSF cytokine levels were determined during acute meningitis. Cortical damage and hippocampal apoptosis were assessed 42 h after infection. Morris water maze and auditory brainstem responses were used to assess neurofunctional outcome 3 weeks after infection.ResultsWe found significantly reduced apoptosis in the hippocampal subgranular zone in infant rats receiving adjuvant Trocade (p < 0.01) or combined adjuvant therapy (p < 0.001). Cortical necrosis was significantly reduced in rats treated with adjuvant daptomycin (p < 0.05) or combined adjuvant therapy (p < 0.05) compared to ceftriaxone monotherapy. Six hours after treatment initiation, CSF cytokine levels were significantly reduced for TNF-α (p < 0.01), IL-1β (p < 0.01), IL-6 (p < 0.001) and IL-10 (p < 0.01) in animals receiving combined adjuvant intervention compared to ceftriaxone monotherapy. Importantly, combined adjuvant therapy significantly improved learning and memory performance in infected animals and reduced hearing loss (77.14 dB vs 60.92 dB, p < 0.05) by preserving low frequency hearing capacity, compared to ceftriaxone monotherapy.ConclusionCombined adjuvant therapy with the non-bacteriolytic antibiotic daptomycin and the MMP inhibitor Trocade integrates the neuroprotective effects of both single adjuvants in experimental paediatric pneumococcal meningitis by reducing neuroinflammation and brain damage, thereby improving neurofunctional outcome. This strategy represents a promising therapeutic option to improve the outcome of paediatric patients suffering from pneumococcal meningitis.

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The peripheral hearing process taking place in the cochlea mainly depends on two distinct sensory cell types: the mechanosensitive hair cells and the spiral ganglion neurons (SGNs). The first respond to the mechanical stimulation exerted by sound pressure waves on their hair bundles by releasing neurotransmitters and thereby activating the latter. Loss of these sensorineural cells is associated with permanent hearing loss. Stem cell-based approaches aiming at cell replacement or in vitro drug testing to identify potential ototoxic, otoprotective, or regenerative compounds have lately gained attention as putative therapeutic strategies for hearing loss. Nevertheless, they rely on efficient and reliable protocols for the in vitro generation of cochlear sensory cells for their implementation. To this end, we have developed a differentiation protocol based on organoid culture systems, which mimics the most important steps of in vivo otic development, robustly guiding mouse embryonic stem cells (mESCs) toward otic sensory neurons (OSNs). The stepwise differentiation of mESCs toward ectoderm was initiated using a quick aggregation method in presence of Matrigel in serum-free conditions. Non-neural ectoderm was induced via activation of bone morphogenetic protein (BMP) signaling and concomitant inhibition of transforming growth factor beta (TGFβ) signaling to prevent mesendoderm induction. Preplacodal and otic placode ectoderm was further induced by inhibition of BMP signaling and addition of fibroblast growth factor 2 (FGF2). Delamination and differentiation of SGNs was initiated by plating of the organoids on a 2D Matrigel-coated substrate. Supplementation with brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) was used for further maturation until 15 days of in vitro differentiation. A large population of neurons with a clear bipolar morphology and functional excitability was derived from these cultures. Immunostaining and gene expression analysis performed at different time points confirmed the transition trough the otic lineage and final expression of the key OSN markers. Moreover, the stem cell-derived OSNs exhibited functional electrophysiological properties of native SGNs. Our established in vitro model of OSNs development can be used for basic developmental studies, for drug screening or for the exploration of their regenerative potential.

Due to the lack of regenerative capacity of the mammalian auditory epithelium, sensory hair cell loss results in permanent hearing deficit. Nevertheless, a population of tissue resident stem/progenitor cells has been recently described. Identification of methods to trigger their activity could lead to exploitation of their potential therapeutically. Here we validate the use of transgenic mice reporting cell cycle progression (FUCCI), and stemness (Lgr5-GFP), as a valuable tool to identify regulators of cell cycle re-entry of supporting cells within the auditory epithelium. The small molecule compound CHIR99021 was used to inhibit GSK3 activity. This led to a significant increase in the fraction of proliferating sphere-forming cells, labeled by the FUCCI markers and in the percentage of Lgr5-GFP + cells, as well as a selective increase in the fraction of S-G2-M cells in the Lgr5 + population. Using whole mount cultures of the organ of Corti we detected a statistically significant increment in the fraction of proliferating Sox2 supporting cells after CHIR99021 treatment, but only rarely appearance of novel MyoVIIa+/Edu + hair cells. In conclusion, these tools provide a robust mean to identify novel regulators of auditory organ regeneration and to clarify the contribution of stem cell activity.

Despite appropriate antibiotic therapy, pneumococcal meningitis (PM) is associated with a case fatality rate of up to 30% in high-income countries. Survivors often suffer from severe lifelong disabilities. An excessive inflammatory reaction drives the pathophysiology, leading to brain damage and neurologic sequelae. We aimed to improve the outcome of experimental PM by simultaneously targeting different pathophysiological mechanisms with combined adjunctive therapies previously shown to be neuroprotective. In vitro, the anti-inflammatory effects of doxycycline and daptomycin were evaluated on primary rat astroglial cells stimulated with Streptococcus pneumoniae. Eleven-day-old infant Wistar rats were infected intracisternally with S. pneumoniae and randomized for treatment with ceftriaxone or combination adjuvant therapy consisting of ceftriaxone, daptomycin, and doxycycline. During acute PM, combined-adjuvant therapy with ceftriaxone, daptomycin, and doxycycline increased the survival rate from 64.1% to 85.8% (P < 0.01) and alleviated weight loss compared to ceftriaxone monotherapy (P < 0.01). Levels of inflammatory cytokines were significantly reduced by combined-adjuvant therapy in vitro (P < 0.0001) and in cerebrospinal fluid in vivo (P < 0.05). In infected animals treated with combined adjunctive therapy, cortical damage was significantly reduced (P < 0.05), and animals showed a trend toward better hearing capacity 3 weeks after the infection (P = 0.089), an effect which was significant in mildly infected animals (48 decibels [dB] versus 67.22 dB; P < 0.05). These mildly infected animals showed significantly reduced cochlear fibrous occlusion (P < 0.01). By combining nonbacteriolytic daptomycin and anti-inflammatory doxycycline with ceftriaxone, the previously reported beneficial effects of the drugs were cumulated and identified the triple-antibiotic therapy as a promising therapeutic option for pediatric PM.

Hearing loss remains the most common long-term complication of pneumococcal meningitis (PM) reported in up to 30% of survivors. Streptococcus pneumoniae have been shown to possess different ototoxic properties. Here we present a novel ex vivo experimental setup to examine in detail the pattern of hair cell loss upon exposure to different S. pneumoniae strains, therefore recapitulating pathogen derived aspects of PM-induced hearing loss. Our results show a higher susceptibility towards S. pneumoniae-induced cochlear damage for outer hair cells (OHC) compared to inner hair cells (IHC), which is consistent with in vivo data. S. pneumoniae-induced hair cell loss was both time and dose-dependent. Moreover, we have found significant differences in the level of cell damage between tissue from the basal and the apical turns. This shows that the higher vulnerability of hair cells located at high frequency regions observed in vivo cannot be explained solely by the spatial organisation and bacterial infiltration from the basal portion of the cochlea. Using a wild type D39 strain and a mutant defective for the pneumolysin (PLY) gene, we also have shown that the toxin PLY is an important factor involved in ototoxic damages. The obtained results indicate that PLY can cause both IHC and OHC loss. Finally, we are reporting here for the first time a higher vulnerability of HC located at the basal and middle cochlear region to pneumolysin-induced damage. The detailed description of the susceptibility of hair cells to Streptococcus pneumoniae provided in this report can in the future determine the choice and the development of novel otoprotective therapies during pneumococcal meningitis.