A single-cell level comparison of human inner ear organoids with the human cochlea and vestibular organs.

Inner ear disorders are among the most common congenital abnormalities; however, current tissue culture models lack the cell type diversity to study these disorders and normal otic development. Here, we demonstrate the robustness of human pluripotent stem cell-derived inner ear organoids (IEOs) and evaluate cell type heterogeneity by single-cell transcriptomics. To validate our findings, we construct a single-cell atlas of human fetal and adult inner ear tissue. Our study identifies various cell types in the IEOs including periotic mesenchyme, type I and type II vestibular hair cells, and developing vestibular and cochlear epithelium. Many genes linked to congenital inner ear dysfunction are confirmed to be expressed in these cell types. Additional cell-cell communication analysis within IEOs and fetal tissue highlights the role of endothelial cells on the developing sensory epithelium. These findings provide insights into this organoid model and its potential applications in studying inner ear development and disorders.

Efficient Viral Transduction in Fetal and Adult Human Inner Ear Explants with AAV9-PHP.B Vectors.

Numerous studies have shown the recovery of auditory function in mouse models of genetic hearing loss following AAV gene therapy, yet translation to the clinic has not yet been demonstrated. One limitation has been the lack of human inner ear cell lines or tissues for validating viral gene therapies. Cultured human inner ear tissue could help confirm viral tropism and efficacy for driving exogenous gene expression in targeted cell types, establish promoter efficacy and perhaps selectivity for targeted cells, confirm the expression of therapeutic constructs and the subcellular localization of therapeutic proteins, and address the potential cellular toxicity of vectors or exogenous constructs. To begin to address these questions, we developed an explant culture method using native human inner ear tissue excised at either fetal or adult stages. Inner ear sensory epithelia were cultured for four days and exposed to vectors encoding enhanced green fluorescent protein (eGFP). We focused on the synthetic AAV9-PHP.B capsid, which has been demonstrated to be efficient for driving eGFP expression in the sensory hair cells of mouse and non-human primate inner ears. We report that AAV9-PHP.B also drives eGFP expression in fetal cochlear hair cells and in fetal and adult vestibular hair cells in explants of human inner ear sensory epithelia, which suggests that both the experimental paradigm and the viral capsid may be valuable for translation to clinical application.

Strontium isotope ratios related to childhood mobility: Revisiting sampling strategies of the calcined human pars petrosa ossis temporalis.

RATIONALE: Strontium isotope analysis can be applied to the calcined human otic capsule in the petrous part (pars petrosa ossis temporalis; PP) to gain information on childhood mobility in archaeological and forensic contexts. However, only a thin layer of the otic capsule, the inner cortex, demonstrates virtually no remodelling. This paper proposes an improved sampling method for the accurate sampling of the inner cortex of the otic capsule to ensure that 87 Sr/86 Sr ratios related to early childhood are obtained. METHODS: Calcined rib and diaphyseal fragments and PP from ten cremation deposits are sampled for strontium isotope analysis, whereby our improved sampling strategy is applied to sample the inner cortex of the otic capsule. This allows inter- and intraskeletal 87 Sr/86 Sr comparison within an Iron Age collection from Oss, The Netherlands. RESULTS: Forty percent (4/10) of the calcined PP that were evaluated for this study show marked differences in 87 Sr/86 Sr (0.00035-0.00065) between the inner cortex and the bone sample surrounding this layer, the external cortex that has higher remodelling rates. Differences in 87 Sr/86 Sr between various skeletal elements also aided in the identification of the minimum number of individuals. CONCLUSIONS: Our study demonstrates the problematic nature of the external cortex and stresses the need for a precise sampling method of the correct areas of the otic capsule. This can only be obtained by cutting the calcined PP midmodiolarly to enable adequate combustion degree assessment, and the correct identification and sampling of the inner cortex of the otic capsule.

Migration and fate of vestibular melanocytes during the development of the human inner ear.

Melanocytes are present in various parts of the inner ear, including the stria vascularis in the cochlea and the dark cell areas in the vestibular organs, where they contribute to endolymph homeostasis. Developmental studies describing the distribution of vestibular melanocytes are scarce, especially in humans. In this study, we investigated the distribution and maturation of the vestibular melanocytes in relation to the developing dark cell epithelium in inner ear specimens from week 5 to week 14 of development and in surgical specimens of the adult ampulla. Vestibular melanocytes were located around the utricle and the ampullae of the semicircular canals before week 7 and were first seen underneath the transitional zones and dark cell areas between week 8 and week 10. At week 10, melanocytes made intimate contact with epithelial cells, interrupting the local basement membrane with their dendritic processes. At week 11, most melanocytes were positioned under the dark cell epithelia. No melanocytes were seen around or in the saccule during all investigated developmental stages. The dark cell areas gradually matured and showed an adult immunohistochemical profile of the characteristic ion transporter protein Na+ /K+ -ATPase α1 by week 14. Furthermore, we investigated the expression of the migration-related proteins ECAD, PCAD, KIT, and KITLG in melanocytes and dark cell epithelium. This is the first study to describe the spatiotemporal distribution of vestibular melanocytes during the human development and thereby contributes to understanding normal vestibular function and pathophysiological mechanisms underlying vestibular disorders.

Imaging Bioluminescent Exogenous Stem Cells in the Intact Guinea Pig Cochlea.

Stem-cell-based therapy may be used to replace damaged or lost neurons in the cochlear nerve of patients suffering from severe-to-profound sensorineural hearing loss. In order to achieve functional recovery in future clinical trials, knowledge about survival of grafted cells and their differentiation into functional neurons is a prerequisite. This calls for non-invasive in vivo visualization of cells and long-term monitoring of their survival and fate after cochlear transplantation. We have investigated if molecular optical imaging enables visualization of exogenous cells in the intact cochlea of guinea pig cadaver heads. Transduced (stem) cells, stably co-expressing fluorescent (copGFP) and bioluminescent (Luc2) reporter molecules, were injected into the internal auditory meatus or directly into the cochlea through the round window. After injection of the cells into the internal auditory meatus, a bright bioluminescent signal was observed in the cavum conchae of the auricle, indicating that light generated by Luc2 is passing through the tympanic membrane and the external auditory meatus. Similar results were obtained after injection of the cells through the round window membrane, either directly into the scala tympani or in Rosenthal's canal within the modiolus of the basal cochlear turn. Imaging of the auditory bulla demonstrated that the bioluminescent signal passes through the tympanic membrane and crevices in the bony wall of the bulla. After opening the auditory bulla, the bioluminescent signal was emanating from the round window. This is the first study demonstrating that bioluminescence imaging enables visualization of luciferase-expressing cells injected into the intact guinea pig cochlea. Anat Rec, 303:427-440, 2020. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

Ouabain Does Not Induce Selective Spiral Ganglion Cell Degeneration in Guinea Pigs.

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.

Neuronal differentiation of hair-follicle-bulge-derived stem cells co-cultured with mouse cochlear modiolus explants.

Stem-cell-based repair of auditory neurons may represent an attractive therapeutic option to restore sensorineural hearing loss. Hair-follicle-bulge-derived stem cells (HFBSCs) are promising candidates for this type of therapy, because they (1) have migratory properties, enabling migration after transplantation, (2) can differentiate into sensory neurons and glial cells, and (3) can easily be harvested in relatively high numbers. However, HFBSCs have never been used for this purpose. We hypothesized that HFBSCs can be used for cell-based repair of the auditory nerve and we have examined their migration and incorporation into cochlear modiolus explants and their subsequent differentiation. Modiolus explants obtained from adult wild-type mice were cultured in the presence of EF1α-copGFP-transduced HFBSCs, constitutively expressing copepod green fluorescent protein (copGFP). Also, modiolus explants without hair cells were co-cultured with DCX-copGFP-transduced HFBSCs, which demonstrate copGFP upon doublecortin expression during neuronal differentiation. Velocity of HFBSC migration towards modiolus explants was calculated, and after two weeks, co-cultures were fixed and processed for immunohistochemical staining. EF1α-copGFP HFBSC migration velocity was fast: 80.5 ± 6.1 µm/h. After arrival in the explant, the cells formed a fascicular pattern and changed their phenotype into an ATOH1-positive neuronal cell type. DCX-copGFP HFBSCs became green-fluorescent after integration into the explants, confirming neuronal differentiation of the cells. These results show that HFBSC-derived neuronal progenitors are migratory and can integrate into cochlear modiolus explants, while adapting their phenotype depending on this micro-environment. Thus, HFBSCs show potential to be employed in cell-based therapies for auditory nerve repair.

Lentiviral transduction and subsequent loading with nanoparticles do not affect cell viability and proliferation in hair-follicle-bulge-derived stem cells in vitro.

The application of stem cells in the treatment of various degenerative diseases is highly promising. However, cell-based therapy could be limited by the problem of low viability of grafted cells and uncertainty about their fate. The combination of molecular imaging and contrast-enhanced MRI may give more insight into the survival and behavior of grafted stem cells. We explore hair-follicle-bulge-derived stem cells (HFBSCs) as a potential candidate for autologous cell-based therapy. HFBSCs are transduced with a lentiviral construct with genes coding for bioluminescent (Luc2) and fluorescent (copGFP) reporter proteins, and subsequently loaded with magnetic nanoparticles to enable MRI visualization. Thus, we investigate for the first time if lentiviral transduction and cellular loading with nanoparticles have a cytotoxic effect upon these stem cells. Transduction efficiency, proliferation rate, cell viability and reporter protein co-expression during long-term culture of transduced HFBSCs were studied using fluorescence and bioluminescence microscopy. In addition, the effect of TMSR50 nanoparticles on proliferation and viability was investigated using the MTS assay and bioluminescence microscopy. The amount of TMSR50-loaded HFBSCs needed to reach signal threshold for MRI was assessed using an agarose phantom. Transduction with the Luc2-copGFP construct did not influence senescence, proliferation, doubling time, and differentiation of the HFBSCs. CopGFP expression was visible immediately after transduction and persisted for at least 15 passages, concomitantly with Luc2 expression. Cellular loading with TMSR50 nanoparticles did not affect cell viability and proliferation. The results imply that combined MRI and bioluminescence imaging may enable in vivo localization and long-term monitoring of grafted viable HFBSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Development of the stria vascularis and potassium regulation in the human fetal cochlea: Insights into hereditary sensorineural hearing loss.

Sensorineural hearing loss (SNHL) is one of the most common congenital disorders in humans, afflicting one in every thousand newborns. The majority is of heritable origin and can be divided in syndromic and nonsyndromic forms. Knowledge of the expression profile of affected genes in the human fetal cochlea is limited, and as many of the gene mutations causing SNHL likely affect the stria vascularis or cochlear potassium homeostasis (both essential to hearing), a better insight into the embryological development of this organ is needed to understand SNHL etiologies. We present an investigation on the development of the stria vascularis in the human fetal cochlea between 9 and 18 weeks of gestation (W9-W18) and show the cochlear expression dynamics of key potassium-regulating proteins. At W12, MITF+/SOX10+/KIT+ neural-crest-derived melanocytes migrated into the cochlea and penetrated the basement membrane of the lateral wall epithelium, developing into the intermediate cells of the stria vascularis. These melanocytes tightly integrated with Na+/K+-ATPase-positive marginal cells, which started to express KCNQ1 in their apical membrane at W16. At W18, KCNJ10 and gap junction proteins GJB2/CX26 and GJB6/CX30 were expressed in the cells in the outer sulcus, but not in the spiral ligament. Finally, we investigated GJA1/CX43 and GJE1/CX23 expression, and suggest that GJE1 presents a potential new SNHL associated locus. Our study helps to better understand human cochlear development, provides more insight into multiple forms of hereditary SNHL, and suggests that human hearing does not commence before the third trimester of pregnancy.

Distribution and development of peripheral glial cells in the human fetal cochlea.

The adult human cochlea contains various types of peripheral glial cells that envelop or myelinate the three different domains of the spiral ganglion neurons: the central processes in the cochlear nerve, the cell bodies in the spiral ganglia, and the peripheral processes in the osseous spiral lamina. Little is known about the distribution, lineage separation and maturation of these peripheral glial cells in the human fetal cochlea. In the current study, we observed peripheral glial cells expressing SOX10, SOX9 and S100B as early as 9 weeks of gestation (W9) in all three neuronal domains. We propose that these cells are the common precursor to both mature Schwann cells and satellite glial cells. Additionally, the peripheral glial cells located along the peripheral processes expressed NGFR, indicating a phenotype distinct from the peripheral glial cells located along the central processes. From W12, the spiral ganglion was gradually populated by satellite glial cells in a spatiotemporal gradient. In the cochlear nerve, radial sorting was accomplished by W22 and myelination started prior to myelination of the peripheral processes. The developmental dynamics of the peripheral glial cells in the human fetal cochlea is in support of a neural crest origin. Our study provides the first overview of the distribution and maturation of peripheral glial cells in the human fetal cochlea from W9 to W22.

Neurosensory development and cell fate determination in the human cochlea.

BACKGROUND: Hearing depends on correct functioning of the cochlear hair cells, and their innervation by spiral ganglion neurons. Most of the insight into the embryological and molecular development of this sensory system has been derived from animal studies. In contrast, little is known about the molecular expression patterns and dynamics of signaling molecules during normal fetal development of the human cochlea. In this study, we investigated the onset of hair cell differentiation and innervation in the human fetal cochlea at various stages of development. RESULTS: At 10 weeks of gestation, we observed a prosensory domain expressing SOX2 and SOX9/SOX10 within the cochlear duct epithelium. In this domain, hair cell differentiation was consistently present from 12 weeks, coinciding with downregulation of SOX9/SOX10, to be followed several weeks later by downregulation of SOX2. Outgrowing neurites from spiral ganglion neurons were found penetrating into the cochlear duct epithelium prior to hair cell differentiation, and directly targeted the hair cells as they developed. Ubiquitous Peripherin expression by spiral ganglion neurons gradually diminished and became restricted to the type II spiral ganglion neurons by 18 weeks. At 20 weeks, when the onset of human hearing is thought to take place, the expression profiles in hair cells and spiral ganglion neurons matched the expression patterns of the adult mammalian cochleae. CONCLUSIONS: Our study provides new insights into the fetal development of the human cochlea, contributing to our understanding of deafness and to the development of new therapeutic strategies to restore hearing.

Class III ß-tubulin, a novel biomarker in the human melanocyte lineage.

It is generally thought that class III ß-tubulin expression is limited to cells of the neural lineage and is therefore often used to identify neurons amongst other cell types, both in vivo and in vitro. Melanocytes are derived from the neural crest and share both morphological features and functional characteristics with peripheral neurons. Here, we show that these similarities extend to class III ß-tubulin (TUBB3) expression, and that human melanocytes express this protein both in vivo and in vitro. In addition, we studied the expression of class III ß-tubulin in two murine melanogenic cell lines and show that expression of this protein starts as melanoblasts mature into melanocytes. Melanin bleaching experiments revealed close proximity between melanin and TUBB3 proteins. In vitro stimulation of primary human melanocytes by α-MSH indicated separate regulatory mechanisms for melanogenesis and to TUBB3 expression. Together, these observations imply that human melanocytes express TUBB3 and that this protein should be recognized as a wider marker for multiple neural crest-derived cells.

The role of electrophonics in electroacoustic stimulation of the guinea pig cochlea.

HYPOTHESIS: Interactions between cochlear responses to combined electrical and acoustic stimulation (EAS) depend on electrically evoked hair cell activity (i.e., electrophonics). BACKGROUND: Although relevant for EAS strategies in cochlear implant users with residual low-frequency hearing, cochlear responses to EAS are not well characterized. Previously, we have shown that acoustically evoked compound action potentials (CAPs) can be suppressed by electrical stimulation. In the present study, we characterized the role of electrophonics in CAP suppression in guinea pigs, under conditions representative of clinically applied EAS. METHODS: Electrophonics depend on the frequency spectrum of the electric pulse train, which is mainly determined by pulse width and, to a lesser extent, by pulse rate. We measured suppression of tone-evoked CAPs by electric pulse trains, while varying the pulse width (80 - 400 µs, n = 5) and the pulse rate (500 - 4000 pps, n = 5). The role of outer hair cells (OHCs) in electrophonics was tested in animals with varying degrees of OHC loss (n = 24). RESULTS: Suppression of acoustically evoked CAPs varied with pulse width, indicating that electrophonics were involved. Short pulse widths resulted in minimal CAP suppression at low acoustic frequencies. Pulse rate did not significantly affect CAP suppression. OHC loss had no significant effect on electrophonic activity. CONCLUSION: Electrophonic activity was present in cochleae with extensive basal hair cell loss, indicating that electrophonics can occur in EAS users. Our results show that short pulse widths are optimal for use in EAS stimulation strategies, on the assumption that minimal suppression is best.

Spiral ganglion cell morphology in guinea pigs after deafening and neurotrophic treatment.

It is well known that spiral ganglion cells (SGCs) degenerate in hair-cell-depleted cochleas and that treatment with exogenous neurotrophins can prevent this degeneration. Several studies reported that, in addition, SGC size decreases after deafening and increases after neurotrophic treatment. The dynamics of these cell size changes are not well known. In a first experiment we measured size, shape (circularity) and intracellular density of SGCs in guinea pigs at various moments after deafening (1, 2, 4, 6, and 8 weeks) and at various cochlear locations. In a second experiment, the effect of treatment with brain-derived neurotrophic factor (BDNF) on SGC morphology was investigated at various cochlear locations in deafened guinea pigs. We found that SGC size gradually decreased after deafening in the basal and middle cochlear turns. Already after one week a decrease in size was observed, which was well before the number of SGCs started to decrease. After BDNF treatment SGCs became noticeably larger than normal throughout the cochlea, including the middle and apical turns, whereas an effect on survival of SGCs was primarily observed in the basal turn. Thus, both after deafening and after neurotrophic treatment a change in size occurs before survival is affected. Morphological changes were not restricted to a subpopulation of SGCs. We argue that although changes in cell size and changes in survival might be manifestations of two separate mechanisms, morphological measures such as size, circularity and intracellular density are indicative for survival and degeneration.

Combined administration of kanamycin and furosemide does not result in loss of vestibular function in Guinea pigs.

Aminoglycoside antibiotics are known to damage the vestibular and auditory sensory epithelia. Although loop diuretics enhance the cochleotoxic effect of aminoglycosides, it is not known whether concomitant administration of an aminoglycoside and a loop diuretic affects the vestibular system. The aim of our study was to investigate the effect of co-administration of kanamycin and furosemide upon the otolith organs and to compare it to the known vestibulotoxic effect of gentamicin. Five guinea pigs were injected with a single dose of both kanamycin (400 mg/kg, s.c.) and furosemide (100 mg/kg, i.v.), 5 animals received gentamicin (100 mg/kg, i.p.) for 10 days, and 5 untreated animals served as controls. After 7 days, vestibular function was assessed by measuring vestibular short-latency evoked potentials (VsEPs) to linear acceleration stimuli and cochlear function by auditory brainstem responses (ABRs) to clicks. Hair cell densities were determined in phalloidin-stained whole mounts of the utricles and saccules, and in midmodiolar sections of resin-embedded cochleae. Co-administration of kanamycin and furosemide had no significant effect on VsEPs and hair cell densities in the utricles and saccules were not reduced. ABR thresholds were increased to a great extent (by ∼60 dB), and histologically a severe loss of cochlear hair cells was observed. The effect of gentamicin, both on vestibular and cochlear function, was just the opposite. VsEP thresholds to horizontal stimulation were elevated and suprathreshold amplitudes showed a decrease, whereas cochlear function was not reduced. With this protocol, we have a tool to selectively induce cochlear or vestibular damage, which may be of interest to researchers and clinicians alike.

Spiral ganglion cell survival after round window membrane application of brain-derived neurotrophic factor using gelfoam as carrier.

Several studies have shown that treatment with various neurotrophins protects spiral ganglion cells (SGCs) from degeneration in hair-cell deprived cochleas. In most of these studies the neurotrophins are delivered by means of intracochlear delivery methods. Recently, other application methods that might be more suited in cochlear implant patients have been developed. We have examined if round window membrane application of gelfoam infiltrated with a neurotrophin resulted in SGC survival in deafened guinea pigs. Two weeks after deafening, gelfoam cubes infiltrated with 6 µg of brain-derived neurotrophic factor (BDNF) were deposited onto the round window membrane of the right cochleas. Electric pulses were delivered through an electrode positioned within the round window niche to electrically evoke auditory brainstem responses (eABRs). Two or four weeks after deposition of the gelfoam all cochleas were histologically examined. We found that local BDNF treatment enhances the survival of SGCs in the basal cochlear turn after two and four weeks. The treatment had no effect on SGC size or shape. In animals treated with BDNF, eABR amplitudes were smaller than in normal-hearing control animals and similar to those in deafened controls. We conclude that BDNF delivered by means of local gelfoam application provides a protective effect, which is limited compared to intracochlear delivery methods.

Chronic electrical stimulation does not prevent spiral ganglion cell degeneration in deafened guinea pigs.

Several studies have demonstrated that treatment with intracochlear chronic electrical stimulation (CES) protects spiral ganglion cells (SGCs) from degeneration in deafened animals. Other studies could not confirm this effect of CES. The present study examined whether CES in a mode as presented in cochlear implant users (amplitude modulated, high pulse rate) affects survival, morphology and functionality of SGCs in deafened guinea pigs. Eleven guinea pigs were implanted in the right cochlea with an electrode array to monitor the electrically evoked auditory brainstem responses (eABRs). The guinea pigs were deafened four weeks later. Two days after deafening, monopolar CES was started in five animals through three electrodes in the basal cochlear turn. CES lasted 4 hours per day, five days per week, for six weeks. SGC packing densities, perikaryal area, cell circularity, amplitudes of suprathreshold eABRs and eABR thresholds were not affected by CES. SGCs of all implanted cochleae were larger and more circular than SGCs in unimplanted cochleae, but this did not depend on CES treatment. Interestingly, an increase in eABR latencies observed after deafening, occurred faster in CES-treated than in untreated animals. In conclusion, amplitude-modulated chronic electrical stimulation with a high pulse rate does not affect survival, morphology and functionality of spiral ganglion cells with the exception of eABR latencies.

Enhanced survival of spiral ganglion cells after cessation of treatment with brain-derived neurotrophic factor in deafened guinea pigs.

Exogenous delivery of neurotrophic factors into the cochlea of deafened animals rescues spiral ganglion cells (SGCs) from degeneration. To be clinically relevant for human cochlear implant candidates, the protective effect of neurotrophins should persist after cessation of treatment and the treated SGCs should remain functional. In this study, the survival and functionality of SGCs were investigated after temporary treatment with brain-derived neurotrophic factor (BDNF). Guinea pigs in the experimental group were deafened, and 2 weeks later, the right cochleae were implanted with an electrode array and drug delivery cannula. BDNF was administered to the implanted cochleae during a 4-week period via a mini-osmotic pump. After completion of the treatment, the osmotic pumps were removed. Two weeks later, the animals were killed and the survival of SGCs was analyzed. To monitor the functionality of the auditory nerve, electrically evoked auditory brainstem responses (eABRs) were recorded in awake animals throughout the experiment. BDNF treatment resulted in enhanced survival of SGCs 2 weeks after cessation of the treatment and prevented the decreases in size and circularity that are seen in the untreated contralateral cochleae. The amplitude of the suprathreshold eABR response in BDNF-treated animals was significantly larger than in deafened control animals and comparable to that in normal-hearing control animals. The amplitude in the BDNF-treated group did not decrease significantly after cessation of treatment. The eABR latency in BDNF-treated animals was longer than normal and comparable to that in deafened control animals. These morphological and functional findings demonstrate that neurotrophic intervention had a lasting effect, which is promising for future clinical application of neurotrophic factors in implanted human cochleae.

Morphological changes in spiral ganglion cells after intracochlear application of brain-derived neurotrophic factor in deafened guinea pigs.

When guinea pigs are deafened with ototoxic drugs spiral ganglion cells (SGCs) degenerate progressively. Application of neurotrophins can prevent this process. Morphological changes of rescued SGCs have not been quantitatively determined yet. It might be that SGCs treated with neurotrophins are more vulnerable than SGCs in cochleae of normal-hearing guinea pigs. Therefore, the mitochondria and myelinisation of type-I SGCs were studied and the perikaryal area, cell circularity and electron density were determined. Guinea pigs were deafened with a subcutaneous injection of kanamycin followed by intravenous infusion of furosemide. Brain-derived neurotrophic factor (BDNF) delivery was started two weeks after the deafening procedure and continued for four weeks. Four cohorts of cochleae were studied: (1) cochleae of normal-hearing guinea pigs; (2) of guinea pigs two weeks after deafening; (3) six weeks after deafening; (4) cochleae treated with BDNF after deafening. The deafening procedure resulted in a progressive loss of SGCs. Six weeks after deafening the size of mitochondria, perikaryal area and cell circularity of the remaining untreated SGCs were decreased and the number of layers of the myelin sheath was reduced. In the basal part of the cochlea BDNF treatment rescued SGCs from degeneration. SGCs treated with BDNF were larger than SGCs in normal-hearing guinea pigs, whereas circularity had normal values and electron density was unchanged. The number of layers in the myelin sheath of BDNF-treated SGCs was reduced as compared to the number of layers in the myelin sheath of SGCs in normal-hearing guinea pigs. The morphological changes of SGCs might be related to the rapid loss of SGCs that has been reported to occur after cessation of BDNF treatment.