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Sensorineural hearing loss is mainly caused by irreversible damage to sensory hair cells (HCs). A subgroup of supporting cells (SCs) in the cochlea express leucine-rich repeat-containing G-protein coupled receptor 5 (LGR5), a marker for tissue-resident stem cells. LGR5+ SCs could be used as an endogenous source of stem cells for regeneration of HCs to treat hearing loss. Here, we report long-term presence of LGR5+ SCs in the mature adult cochlea and survival of LGR5+ SCs after severe ototoxic trauma characterized by partial loss of inner HCs and complete loss of outer HCs. Surviving LGR5+ SCs (confirmed by GFP expression) were located in the third row of Deiters’ cells. We observed a change in the intracellular localization of GFP, from the nucleus in normal-hearing to cytoplasm and membrane in deafened mice. These data suggests that the adult mammalian cochlea possesses properties essential for regeneration even after severe ototoxic trauma.

BackgroundNeuroblastoma is a commonly lethal solid tumor of childhood and intensive chemoradiotherapy treatment cures ~50% of children with high-risk disease. The addition of immunotherapy using dinutuximab, a monoclonal antibody directed against the GD2 disialoganglioside expressed on neuroblasts, improves survival when incorporated into front-line therapy and shows robust activity in regressing relapsed disease when combined with chemotherapy. Still, many children succumb to neuroblastoma despite receiving dinutuximab-based immunotherapy, and efforts to counteract the immune suppressive signals responsible are warranted. Animal models of human cancers provide useful platforms to study immunotherapies. TH-MYCN transgenic mice are immunocompetent and develop neuroblastomas at autochthonous sites due to enforced MYCN expression in developing neural crest tissues. However, GD2-directed immunotherapy in this model has been underutilized due to the prevailing notion that TH-MYCN neuroblasts express insufficient GD2 to be targeted.MethodsTH-MYCN mice were treated with 14G2a (anti-GD2 antibody), isotype antibody, or phosphate buffered saline from day 14 of life until day 100 or signs of morbidity. Survival was recorded, and tumors were isolated in terminal surgeries for analysis of GD2 expression and immune cell frequencies. Tumors from untreated mice were explanted for generation into cell lines, and GD2 expression was recorded with serial passage in tissue culture. Immunocytology and immunoblotting were performed to evaluate for adrenergic and mesenchymal markers of neuroblasts. Survival curves compared using Kaplan-Meier method with a log-rank test for significance. Unpaired two-tailed Student’s t-tests used for comparison of groups in flow cytometry analysis.Results14G2a markedly extends survival in such TH-MYCN mice. Additionally, neuroblasts in 14G2a-treated mice have reduced GD2 expression and fewer macrophage and myeloid-derived suppressor cells in their tumor microenvironments. Neuroblasts in TH-MYCN-driven tumors express GD2 at levels comparable to human neuroblastomas but rapidly lose GD2 expression when explanted ex vivo to establish tumor cell lines. The loss of GD2 expression ex vivo is associated with a transition from an adrenergic to mesenchymal state that is maintained when reimplanted in vivo.ConclusionsOur findings support the utility of the TH-MYCN model to inform GD2-directed immunotherapy approaches for neuroblastoma as well as opportunities to investigate drivers of adrenergic to mesenchymal fate decisions.

Round window membrane (RWM) application of ouabain is known to selectively destroy type I spiral ganglion cells (SGCs) in cochleas of several rodent species, while leaving hair cells intact. This protocol has been used in rats and Mongolian gerbils, but observations in the guinea pig are conflicting. This is why we reinvestigated the effect of ouabain on the guinea pig cochlea. Ouabain solutions of different concentrations were placed, in a piece of gelfoam, upon the RWM of the right cochleas. Auditory function was assessed using acoustically evoked auditory brainstem responses (aABR). Finally, cochleas were fixed and processed for histological examination. Due to variability within treatment groups, histological data was pooled and three categories based upon general histological observations were defined: cochleas without outer hair cell (OHC) and SGC loss (Category 1), cochleas with OHC loss only (Category 2), and cochleas with OHC and SGC loss (Category 3). Animals treated with 1 mM or 10 mM ouabain showed shifts in hearing thresholds, corresponding with varying histological changes in their cochleas. Most cochleas exhibited complete outer hair cell loss in the basal and middle turns, while some had no changes, together with either moderate or near-complete loss of SGCs. Neither loss of inner hair cells nor histological changes of the stria vascularis were observed in any of the animals. Cochleas in Category 1 had normal aABRs and morphology. On average, in Category 2 OHC loss was 46.0±5.7%, SGC loss was below threshold, ABR threshold shift was 44.9±2.7 dB, and ABR wave II amplitude was decreased by 17.1±3.8 dB. In Category 3 OHC loss was 68.3±6.9%, SGC loss was 49.4±4.3%, ABR threshold shift was 39.0±2.4 dB, and ABR amplitude was decreased by 15.8±1.6 dB. Our results show that ouabain does not solely destroy type I SGCs in the guinea pig cochlea.

In mammals, hair cell loss is irreversible and leads to hearing loss. To develop and test the functioning of different strategies aiming at hair cell regeneration, animal models of sensorineural hearing loss are essential. Although cochleae of these animals should lack hair cells, supporting cells should be preserved forming an environment for the regenerated hair cells. In this study, we investigated how ototoxic treatment with kanamycin and furosemide changes the structure of cochlear sensory epithelium in mice. The study also compared different tissue preparation protocols for scanning electron microscopy (SEM). Cochleae were collected from deafened and nondeafened mice and further processed for plastic mid modiolar sections and SEM. For comparing SEM protocols, cochleae from nondeafened mice were processed using three protocols: osmium-thiocarbohydrazide-osmium (OTO), tannic acid-arginine-osmium, and the conventional method with gold-coating. The OTO method demonstrated optimal cochlear tissue preservation. Histological investigation of cochleae of deafened mice revealed that the supporting cells enlarged and ultimately replaced the lost hair cells forming types 1 and 2 phalangeal scars in a base towards apex gradient. The type 3 epithelial scar, flattened epithelium, has not been seen in analysed cochleae. The study concluded that mice deafened with kanamycin and furosemide formed scars containing supporting cells, which renders this mouse model suitable for testing various hair cell regeneration approaches. Microsc. Res. Tech. 79:766-772, 2016. © 2016 Wiley Periodicals, Inc.

Damage to and loss of the organ of Corti leads to secondary degeneration of the spiral ganglion cell (SGC) somata of the auditory nerve. Extensively examined in animal models, this degeneration process of SGC somata following deafening is well known. However, degeneration of auditory nerve axons, which conduct auditory information towards the brainstem, and its relation to SGC soma degeneration are largely unknown. The consequences of degeneration of the axons are relevant for cochlear implantation, which is applied to a deafened system but depends on the condition of the auditory nerve. We investigated the time sequence of degeneration of myelinated type I axons in deafened guinea pigs. Auditory nerves in six normal-hearing and twelve deafened animals, two, six and fourteen weeks (for each group four) after deafening were histologically analyzed. We developed a semi-automated method for axon counting, which allowed for a relatively large sample size (20% of the total cross-sectional area of the auditory nerve). We observed a substantial loss of auditory nerve area (29%), reduction in axon number (59%) and decrease in axoplasm area (41%) fourteen weeks after deafening compared to normal-hearing controls. The correlation between axonal degeneration and that of the SGC somata in the same cochleas was high, although axonal structures appeared to persist longer than the somata, suggesting a slower degeneration process. In the first two weeks after induction of deafness, the axonal cross-sectional area decreased but the axon number did not. In conclusion, the data strongly suggest that each surviving SGC possesses an axon.

Hypothesis How to induce most efficiently severe sensorineural hearing loss in mice using a single coadministration of an aminoglycoside antibiotic and a loop diuretic? Background The coadministration of aminoglycosides and a loop diuretic has been widely used to induce hair cell and spiral ganglion cell loss in guinea pigs. However, the development of new treatment strategies against sensorineural hearing loss, such as tissue engineering techniques, requires the use of mouse models. Previous attempts to induce hearing loss in mice have rendered inconsistent results because of resistance to aminoglycoside-induced ototoxicity. Especially inner hair cells seem to be resistant to aminoglycoside-induced ototoxicity. Methods In the present study, we aim to optimize hearing loss in mice, using a single high-dose kanamycin (700 and 1,000 mg/kg) injection followed by a furosemide (100 mg/kg) administration. Although previous studies used intraperitoneal furosemide injections 30 minutes after kanamycin administration, we used intravenous furosemide injections administered within 5 minutes after kanamycin treatment. Results Auditory brain stem responses illustrated severe threshold shifts, and histologic analysis showed marked outer hair cell destruction as well as spiral ganglion cell loss. The present protocol results in more severe inner hair cell loss when compared with the results of previous researches. Conclusion We conclude that severe sensorineural hearing loss can be induced in mice. Moreover, we found that this mouse model can be augmented via the use of rapid intravenous furosemide administrations to maximize inner hair cell loss.

OBJECTIVE To characterize the effects of deafening and subsequent treatment with brain-derived neurotrophic factor (BDNF) on the peripheral processes (PPs) of spiral ganglion cells (SGCs) in guinea pigs. BACKGROUND BDNF may prevent degeneration of neural structures after loss of hair cells with possible relevance for cochlear implant candidates. METHODS Guinea pigs were deafened with a combination of kanamycin and furosemide. Two weeks after deafening, intracochlear BDNF treatment was started with osmotic pumps for 4 weeks. Two weeks after cessation of BDNF treatment, the cochleae were analyzed. PPs were counted and morphologically characterized with respect to myelination, size, and shape. RESULTS Deafening reduced the number of PPs. We found that BDNF treatment, started 2 weeks after deafening, significantly reduced this degenerative effect. The remaining processes showed an altered morphology; compared with normal, the size was reduced in deafened untreated animals and increased in BDNF-treated animals. The myelin sheath seemed thinner in BDNF-treated animals. CONCLUSION We conclude that BDNF has potential as an agent that can improve the interface between cochlear implants and the auditory periphery.

In the developing cochlea, Wnt/β-catenin signaling positively regulates the proliferation of precursors and promotes the formation of hair cells by up-regulating Atoh1 expression. Not much, however, is known about the regulation of Wnt/β-catenin activity in the cochlea. In multiple tissues, the activity of Wnt/β-catenin signaling is modulated by an interaction between LGR receptors and their ligands from the R-spondin family. The deficiency in Lgr4 and Lgr5 genes leads to developmental malformations and lethality. Using the Lgr5 knock-in mouse line we show that loss of LGR5 function increases Wnt/β-catenin activity in the embryonic cochlea, resulting in a mild overproduction of inner and outer hair cells. Supernumerary hair cells are likely formed due to an up-regulation of the "pro-hair cell" transcription factors Atoh1, Nhlh1, and Pou4f3. Using a hypomorphic Lgr4 mouse model we showed a mild overproduction of outer hair cells in the heterozygous and homozygous Lgr4 mice. The loss of LGR4 function prolonged the proliferation in the mid-basal turn of E13 cochleae, causing an increase in the number of SOX2-positive precursor cells within the pro-sensory domain. The premature differentiation of hair cells progressed in a medial to lateral gradient in Lgr4 deficient embryos. No significant up-regulation of Atoh1 was observed following Lgr4 deletion. Altogether, our findings suggest that LGR4 and LGR5 play an important role in the regulation of hair cell differentiation in the embryonic cochlea.

Cisplatin-induced ototoxicity is correlated with functional and morphological changes in the organ of Corti, the stria vascularis and the spiral ganglion. However, the cochlear sites of cisplatin uptake and accumulation have not been properly identified. Therefore, we have developed an immunohistochemical method to, indirectly, detect cisplatin in semithin cryosections of the guinea pig cochlea (basal turn) using an antiserum containing antibodies against cisplatin-DNA adducts. Platinated DNA was present in the nuclei of most cells in the organ of Corti and the lateral wall after cisplatin administration. Nuclear immunostaining was most pronounced in the outer hair cells, the marginal cells and the spiral ligament fibrocytes. This study is the first to demonstrate the presence of cisplatin in histological sections of the cochlea.

The human melatonin MT1 receptor—belonging to the large family of G protein-coupled receptors (GPCRs)—plays a key role in circadian rhythm regulation and is notably involved in sleep disorders and depression. Structural and functional information at the molecular level are highly desired for fine characterization of this receptor; however, adequate techniques for isolating soluble MT1 material suitable for biochemical and biophysical studies remain lacking. Here we describe the evaluation of a panel of constructs and host systems for the production of recombinant human MT1 receptors, and the screening of different conditions for their solubilization and purification. Our findings resulted in the establishment of an original strategy using a mixture of Fos14 and CHAPS detergents to extract and purify a recombinant human MT1 from Pichia pastoris membranes. This procedure enabled the recovery of relatively pure, monomeric and ligand-binding active MT1 receptor in the near-milligram range. A comparative study based on extensive ligand-binding characterization highlighted a very close correlation between the pharmacological profiles of MT1 purified from yeast and the same receptor present in mammalian cell membranes. The high quality of the purified MT1 was further confirmed by its ability to activate its cognate Gαi protein partner when reconstituted in lipid discs, thus opening novel paths to investigate this receptor by biochemical and biophysical approaches.

Until 1993 it was commonly accepted that regeneration of vestibular hair cells was not possible in mammals. Two histological studies then showed structural evidence for spontaneous regeneration of vestibular hair cells after gentamicin treatment. There is less evidence for functional recovery going along with this regenerative process; in other words, do regenerated hair cells function adequately? This study aims to address this question, and in general evaluates whether spontaneous functional recovery may occur, in the short or long term, in mammals after ototoxic insult. Guinea pigs were treated with gentamicin for 10 consecutive days at a daily dose of 125 mg/kg body weight. Survival times varied from 1 day to 16 weeks. Vestibular short-latency evoked potentials (VsEPs) to linear acceleration pulses were recorded longitudinally to assess otolith function. After the final functional measurements we performed immunofluorescence histology for hair cell counts. Auditory brainstem responses (ABRs) to click stimuli were recorded to assess cochlear function. As intended, gentamicin treatment resulted in significant loss of utricular hair cells and accompanying declines in VsEPs. Hair cell counts 8 or 16 weeks after treatment did not significantly differ from counts after shorter survival periods. Maximal functional loss was achieved 1-4 weeks after treatment. After this period, only 2 animals showed recovery of VsEP amplitude - all other animals did not reveal signs of regeneration or recovery. In contrast, after initial ABR threshold shifts there was a small but significant recovery. We conclude that spontaneous recovery of otolith function, in contrast to cochlear function, is very limited in guinea pigs. These results support the concept of intratympanic gentamicin treatment where gentamicin is used for chemoablation of the vestibular sensory epithelia.

In order to establish the more precise localization of IFPs in the vestibular labyrinth we have developed an immunohistochemical method using semithin cryosections from guinea pig inner ear. The vestibular end organs were fixed by intralabyrinthine perfusion with formaldehyde and glutaraldehyde. Microdissection was performed, followed by decalcification in EDTA. After specimen embedding in gelatin, semithin cryosections (1 micron) were prepared. Prior to the immunohistochemical staining, a new antigen-unmasking treatment was performed. Monoclonal antibodies for cytokeratins (Cks), vimentin, neurofilament protein (NF), and glial fibrillary acidic protein (GFAP) were used and visualized with an indirect ABC method. In the guinea pig vestibular labyrinth, Cks8, 18 and 19 were present. Vimentin and NF were stained positively, GFAP negatively. These results are in accordance with our previous results in the human vestibular labyrinth except for Ck7. The high-resolution cryotechnique in combination with a new antigen-unmasking method may yield more, and more detailed information about the localization of IFPs in the vestibular region.

Chronic recurrent parotitis (CRP) is recurrent parotid inflammation with non-obstructive sialectasis. Therapies which produce acinar atrophy or remove the acini are effective in treating CRP. Parotidectomy, tympanic neurectomy, duct ligation, and radiation therapy have either a low success rate or a high risk of morbidity. Intraductal antibiotic instillation has been proposed as a possible method of treatment. We hypothesized that the cytotoxic effects of tetracycline could produce acinar atrophy. A double-blind experiment of intraductal tetracycline instillation was performed in ten rabbits. Acinar atrophy and acute inflammation were found in 40% of the tetracycline treated glands; controls had a complete absence of these histologic changes. These results support the use of tetracycline instillation to produce acinar atrophy and therefore, intraductal tetracycline may be an effective, low-risk therapy for CRP. The clinical features of CRP will be reviewed and therapeutic implications discussed.

FGFRs (fibroblast growth factor receptors) are encoded by four genes (FGFR1 – 4). Alternative splicing results in various receptor isoforms. The FGFR2-IIIb variant is present in a wide variety of epithelia, including the bladder epithelium. Recently, we have shown that FGFR2-IIIb is downregulated in a subset of transitional cell carcinomas of the bladder, and that this downregulation is associated with a poor prognosis. We investigated possible tumour suppressive properties of FGFR2-IIIb by transfecting two human bladder tumour cell lines, J82 and T24, which have no endogenous FGFR2-IIIb expression, with FGFR2-IIIb cDNA. No stable clones expressing FGFR2-IIIb were isolated with the J82 cell line. For the T24 cell line, stable transfectants expressing FGFR2-IIIb had reduced growth in vitro and formed fewer tumours in nude mice which, in addition, grew more slowly. The potential mechanisms leading to decreased FGFR2-IIIb mRNA levels were also investigated. The 5′ region of the human FGFR2 gene was isolated and found to contain a CpG island which was partially methylated in more than half the cell lines and tumours which do not express FGFR2-IIIb. No homozygous deletion was identified in any of the tumours or cell lines with reduced levels of FGFR2-IIIb. Mutational analysis of the entire coding region of FGFR2-IIIb at the transcript level was performed in 33 bladder tumours. In addition to normal FGFR2-IIIb mRNA, abnormal transcripts were detected in two tumour samples. These abnormal mRNAs resulted from exon skipping which affected the region encoding the kinase domain. Altogether, these results show that FGFR2-IIIb has tumour growth suppressive properties in bladder carcinomas and suggest possible mechanisms of FGFR2 gene inactivation.