Runx1 controls auditory sensory neuron diversity in mice.

Sound stimulus is encoded in mice by three molecularly and physiologically diverse subtypes of sensory neurons, called Ia, Ib, and Ic spiral ganglion neurons (SGNs). Here, we show that the transcription factor Runx1 controls SGN subtype composition in the murine cochlea. Runx1 is enriched in Ib/Ic precursors by late embryogenesis. Upon the loss of Runx1 from embryonic SGNs, more SGNs take on Ia rather than Ib or Ic identities. This conversion was more complete for genes linked to neuronal function than to connectivity. Accordingly, synapses in the Ib/Ic location acquired Ia properties. Suprathreshold SGN responses to sound were enhanced in Runx1CKO mice, confirming the expansion of neurons with Ia-like functional properties. Runx1 deletion after birth also redirected Ib/Ic SGNs toward Ia identity, indicating that SGN identities are plastic postnatally. Altogether, these findings show that diverse neuronal identities essential for normal auditory stimulus coding arise hierarchically and remain malleable during postnatal development.

Dynamic Heterogeneity Shapes Patterns of Spiral Ganglion Activity.

Neural response properties that typify primary sensory afferents are critical to fully appreciate because they establish and, ultimately represent, the fundamental coding design used for higher-level processing. Studies illuminating the center-surround receptive fields of retinal ganglion cells, for example, were ground-breaking because they determined the foundation of visual form detection. For the auditory system, a basic organizing principle of the spiral ganglion afferents is their extensive electrophysiological heterogeneity establishing diverse intrinsic firing properties in neurons throughout the spiral ganglion. Moreover, these neurons display an impressively large array of neurotransmitter receptor types that are responsive to efferent feedback. Thus, electrophysiological diversity and its neuromodulation are a fundamental encoding mechanism contributed by the primary afferents in the auditory system. To place these features into context, we evaluated the effects of hyperpolarization and cAMP on threshold level as indicators of overall afferent responsiveness in CBA/CaJ mice of either sex. Hyperpolarization modified threshold gradients such that distinct voltage protocols could shift the relationship between sensitivity and stimulus input to reshape resolution. This resulted in an "accordion effect" that appeared to stretch, compress, or maintain responsivity across the gradient of afferent thresholds. cAMP targeted threshold and kinetic shifts to rapidly adapting neurons, thus revealing multiple cochleotopic properties that could potentially be independently regulated. These examples of dynamic heterogeneity in primary auditory afferents not only have the capacity to shift the range, sensitivity, and resolution, but to do so in a coordinated manner that appears to orchestrate changes with a seemingly unlimited repertoire.SIGNIFICANCE STATEMENT How do we discriminate the more nuanced qualities of the sound around us? Beyond the basics of pitch and loudness, aspects, such as pattern, distance, velocity, and location, are all attributes that must be used to encode acoustic sensations effectively. While higher-level processing is required for perception, it would not be unexpected if the primary auditory afferents optimized receptor input to expedite neural encoding. The findings reported herein are consistent with this design. Neuromodulation compressed, expanded, shifted, or realigned intrinsic electrophysiological heterogeneity to alter neuronal responses selectively and dynamically. This suggests that diverse spiral ganglion phenotypes provide a rich substrate to support an almost limitless array of coding strategies within the first neural element of the auditory pathway.

Three-dimensional tonotopic mapping of the human cochlea based on synchrotron radiation phase-contrast imaging.

The human cochlea transforms sound waves into electrical signals in the acoustic nerve fibers with high acuity. This transformation occurs via vibrating anisotropic membranes (basilar and tectorial membranes) and frequency-specific hair cell receptors. Frequency-positions can be mapped within the cochlea to create a tonotopic chart which fits an almost-exponential function with lowest frequencies positioned apically and highest frequencies positioned at the cochlear base (Bekesy 1960, Greenwood 1961). To date, models of frequency positions have been based on a two-dimensional analysis with inaccurate representations of the cochlear hook region. In the present study, the first three-dimensional frequency analysis of the cochlea using dendritic mapping to obtain accurate tonotopic maps of the human basilar membrane/organ of Corti and the spiral ganglion was performed. A novel imaging technique, synchrotron radiation phase-contrast imaging, was used and a spiral ganglion frequency function was estimated by nonlinear least squares fitting a Greenwood-like function (F = A (10ax - K)) to the data. The three-dimensional tonotopic data presented herein has large implications for validating electrode position and creating customized frequency maps for cochlear implant recipients.

Purinergic Signaling Controls Spontaneous Activity in the Auditory System throughout Early Development.

Spontaneous bursts of electrical activity in the developing auditory system arise within the cochlea before hearing onset and propagate through future sound-processing circuits of the brain to promote maturation of auditory neurons. Studies in isolated cochleae revealed that this intrinsically generated activity is initiated by ATP release from inner supporting cells (ISCs), resulting in activation of purinergic autoreceptors, K+ efflux, and subsequent depolarization of inner hair cells. However, it is unknown when this activity emerges or whether different mechanisms induce activity during distinct stages of development. Here we show that spontaneous electrical activity in mouse cochlea from both sexes emerges within ISCs during the late embryonic period, preceding the onset of spontaneous correlated activity in inner hair cells and spiral ganglion neurons, which begins at birth and follows a base to apex developmental gradient. At all developmental ages, pharmacological inhibition of P2Y1 purinergic receptors dramatically reduced spontaneous activity in these three cell types. Moreover, in vivo imaging within the inferior colliculus revealed that auditory neurons within future isofrequency zones exhibit coordinated neural activity at birth. The frequency of these discrete bursts increased progressively during the postnatal prehearing period yet remained dependent on P2RY1. Analysis of mice with disrupted cholinergic signaling in the cochlea indicate that this efferent input modulates, rather than initiates, spontaneous activity before hearing onset. Thus, the auditory system uses a consistent mechanism involving ATP release from ISCs and activation of P2RY1 autoreceptors to elicit coordinated excitation of neurons that will process similar frequencies of sound.SIGNIFICANCE STATEMENT In developing sensory systems, groups of neurons that will process information from similar sensory space exhibit highly correlated electrical activity that is critical for proper maturation and circuit refinement. Defining the period when this activity is present, the mechanisms responsible and the features of this activity are crucial for understanding how spontaneous activity influences circuit development. We show that, from birth to hearing onset, the auditory system relies on a consistent mechanism to elicit correlate firing of neurons that will process similar frequencies of sound. Targeted disruption of this activity will increase our understanding of how these early circuits mature and may provide insight into processes responsible for developmental disorders of the auditory system.

Cochlear supporting cells function as macrophage-like cells and protect audiosensory receptor hair cells from pathogens.

To protect the audiosensory organ from tissue damage from the immune system, the inner ear is separated from the circulating immune system by the blood-labyrinth barrier, which was previously considered an immune-privileged site. Recent studies have shown that macrophages are distributed in the cochlea, especially in the spiral ligament, spiral ganglion, and stria vascularis; however, the direct pathogen defence mechanism used by audiosensory receptor hair cells (HCs) has remained obscure. Here, we show that HCs are protected from pathogens by surrounding accessory supporting cells (SCs) and greater epithelial ridge (GER or Kölliker's organ) cells (GERCs). In isolated murine cochlear sensory epithelium, we established Theiler's murine encephalomyelitis virus, which infected the SCs and GERCs, but very few HCs. The virus-infected SCs produced interferon (IFN)-α/ß, and the viruses efficiently infected the HCs in the IFN-α/ß receptor-null sensory epithelium. Interestingly, the virus-infected SCs and GERCs expressed macrophage marker proteins and were eliminated from the cell layer by cell detachment. Moreover, lipopolysaccharide induced phagocytosis of the SCs without cell detachment, and the SCs phagocytosed the bacteria. These results reveal that SCs function as macrophage-like cells, protect adjacent HCs from pathogens, and provide a novel anti-infection inner ear immune system.

Pleiotrophin increases neurite length and number of spiral ganglion neurons in vitro.

Acoustic trauma, aging, genetic defects or ototoxic drugs are causes for sensorineural hearing loss involving sensory hair cell death and secondary degeneration of spiral ganglion neurons. Auditory implants are the only available therapy for severe to profound sensorineural hearing loss when hearing aids do not provide a sufficient speech discrimination anymore. Neurotrophic factors represent potential therapeutic candidates to improve the performance of cochlear implants (CIs) by the support of spiral ganglion neurons (SGNs). Here, we investigated the effect of pleiotrophin (PTN), a well-described neurotrophic factor for different types of neurons that is expressed in the postnatal mouse cochlea. PTN knockout mice exhibit severe deficits in auditory brainstem responses, which indicates the importance of PTN in inner ear development and function and makes it a promising candidate to support SGNs. Using organotypic explants and dissociated SGN cultures, we investigated the influence of PTN on the number of neurons, neurite number and neurite length. PTN significantly increased the number and neurite length of dissociated SGNs. We further verified the expression of important PTN-associated receptors in the SG. mRNA of anaplastic lymphoma kinase, αv integrin, ß3 integrin, receptor protein tyrosine phosphatase ß/ζ, neuroglycan C, low-density lipoprotein receptor-related protein 1 and syndecan 3 was detected in the inner ear. These results suggest that PTN may be a novel candidate to improve sensorineural hearing loss treatment in the future.

The Estimated Electrode-Neuron Interface in Cochlear Implant Listeners Is Different for Early-Implanted Children and Late-Implanted Adults.

Cochlear implant (CI) programming is similar for all CI users despite limited understanding of the electrode-neuron interface (ENI). The ENI refers to the ability of each CI electrode to effectively stimulate target auditory neurons and is influenced by electrode position, neural health, cochlear geometry, and bone and tissue growth in the cochlea. Hearing history likely affects these variables, suggesting that the efficacy of each channel of stimulation differs between children who were implanted at young ages and adults who lost hearing and received a CI later in life. This study examined whether ENI quality differed between early-implanted children and late-implanted adults. Auditory detection thresholds and most comfortable levels (MCLs) were obtained with monopolar and focused electrode configurations. Channel-to-channel variability and dynamic range were calculated for both types of stimulation. Electrical field imaging data were also acquired to estimate levels of intracochlear resistance. Children exhibited lower average auditory perception thresholds and MCLs compared with adults, particularly with focused stimulation. However, neither dynamic range nor channel-to-channel threshold variability differed between groups, suggesting that children's range of perceptible current was shifted downward. Children also demonstrated increased intracochlear resistance levels relative to the adult group, possibly reflecting greater ossification or tissue growth after CI surgery. These results illustrate physical and perceptual differences related to the ENI of early-implanted children compared with late-implanted adults. Evidence from this study demonstrates a need for further investigation of the ENI in CI users with varying hearing histories.

Insulin-like growth factor 1 promotes cochlear synapse regeneration after excitotoxic trauma in vitro.

In the context of acquired sensorineural hearing loss (SNHL), cochlear hair cells have long been thought to be among the most vulnerable elements in mammalian cochleae. However, recent studies have indicated that the synaptic connection between inner hair cells (IHC) and spiral ganglion neurons (SGN) can be an important target for the treatment of SNHL. Our previous studies in patients with sudden SNHL demonstrated delayed and gradual hearing recovery following topical application of insulin-like growth factor 1 (IGF-1), suggesting that not only protective but also regenerative mechanisms may account for hearing recovery after treatment with IGF-1. We then hypothesized that IGF-1 has the potential to drive the regeneration of IHC-SGN synapses. To test this hypothesis, we investigated the effects of IGF-1 on IHC-SGN synapses using cochlear explant cultures from postnatal day 2 mice that had been damaged by exposure to the excitatory amino acids N-methyl-d-aspartate and kainate. Cochlear explants that lost IHC-SGN synapses upon exposure to excitatory amino acids were cultured with exogenous IGF-1 for an additional 48 h. We observed increased numbers of IHC-SGN synapses after exogenous IGF-1 application. Pharmacological inhibition of the IGF-1 receptor attenuated the restoration of IHC-SGN synapses by exogenous IGF-1. These findings indicated that IGF-1 induces regeneration of IHC-SGN synapses in cochlear explant cultures from postnatal day 2 mice. Therefore, in a future study we will perform in vivo experiments using adult mice to ascertain the effects of IGF-1 on the regeneration of IHC-SGN synapses.

Ancestral Adeno-Associated Virus Vector Delivery of Opsins to Spiral Ganglion Neurons: Implications for Optogenetic Cochlear Implants.

Optogenetics is a transformative technology based on light-sensitive microbial proteins, known as opsins, that enable precise modulation of neuronal activity with pulsed radiant energy. Optogenetics has been proposed as a means to improve auditory implant outcomes by reducing channel interaction and increasing electrode density, but the introduction of opsins into cochlear spiral ganglion neurons (SGNs) in vivo has been challenging. Here we test opsin delivery using a synthetically developed ancestral adeno-associated virus (AAV) vector called Anc80L65. Wild-type C57BL/6 mouse pups were injected via the round window of cochlea with Anc80L65 carrying opsin Chronos under the control of a CAG promoter. Following an incubation of 6-22 weeks, pulsed blue light was delivered to cochlear SGNs via a cochleosotomy approach and flexible optical fiber. Optically evoked auditory brainstem responses (oABRs) and multiunit activity in inferior colliculus (IC) were observed. Post-experiment cochlear histology demonstrated opsin expression in SGNs (mean = 74%), with an even distribution of opsin along the cochlear basal/apical gradient. This study is the first to describe robust SGN transduction, opsin expression, and optically evoked auditory electrophysiology in neonatal mice. Ultimately, this work may provide the basis for a new generation of cochlear implant based on light.

Effect of hyperbaric oxygen on BDNF-release and neuroprotection: Investigations with human mesenchymal stem cells and genetically modified NIH3T3 fibroblasts as putative cell therapeutics.

Hyperbaric oxygen therapy (HBOT) is a noninvasive widely applied treatment that increases the oxygen pressure in tissues. In cochlear implant (CI) research, intracochlear application of neurotrophic factors (NTFs) is able to improve survival of spiral ganglion neurons (SGN) after deafness. Cell-based delivery of NTFs such as brain-derived neurotrophic factor (BDNF) may be realized by cell-coating of the surface of the CI electrode. Human mesenchymal stem cells (MSC) secrete a variety of different neurotrophic factors and may be used for the development of a biohybrid electrode in order to release endogenously-derived neuroprotective factors for the protection of residual SGN and for a guided outgrowth of dendrites in the direction of the CI electrode. HBOT could be used to influence cell behaviour after transplantation to the inner ear. The aim of this study was to investigate the effect of HBOT on the proliferation, BDNF-release and secretion of neuroprotective factors. Thus, model cells (an immortalized fibroblast cell line (NIH3T3)-native and genetically modified) and MSCs were repeatedly (3 x - 10 x) exposed to 100% oxygen at different pressures. The effects of HBO on cell proliferation were investigated in relation to normoxic and normobaric conditions (NOR). Moreover, the neuroprotective and neuroregenerative effects of HBO-treated cells were analysed by cultivation of SGN in conditioned medium. Both, the genetically modified NIH3T3/BDNF and native NIH3T3 fibroblasts, showed a highly significant increased proliferation after five days of HBOT in comparison to normoxic controls. By contrast, the number of MSCs was decreased in MSCs treated with 2.0 bar of HBO. Treating SGN cultures with supernatants of fibroblasts and MSCs significantly increased the survival rate of SGN. HBO treatment did not influence (increase / reduce) this effect. Secretome analysis showed that HBO treatment altered the protein expression pattern in MSCs.

Application of stem cell for the regeneration of spiral ganglion neurons.

Over 278 million of people worldwide suffers from hearing loss, and this disease has significant detrimental effects emotionally and economically on individuals and the society in totality. Treatment using cochlear implant dramatically improve the perception, and production of speech, as well as the patient quality of life, with the different sensorineural hearing loss (SNHL). Yet, there are some challenges faced by a cochlear implant. In this review, we propose the regeneration of spiral ganglion neurons which is an interface neuron using human amniotic fluid mesenchymal stem cells (hAFMSCs), due to its high pluripotency potentials, this stem cell source can regenerate the spiral ganglion and this in-turn will bring back the inner ear hair-cells to functionality.

Genetic variants in the peripheral auditory system significantly affect adult cochlear implant performance.

BACKGROUND: Cochlear implantation is an effective habilitation modality for adults with significant hearing loss. However, post-implant performance is variable. A portion of this variance in outcome can be attributed to clinical factors. Recent physiological studies suggest that the health of the spiral ganglion also impacts post-operative cochlear implant outcomes. The goal of this study was to determine whether genetic factors affecting spiral ganglion neurons may be associated with cochlear implant performance. METHODS: Adults with post-lingual deafness who underwent cochlear implantation at the University of Iowa were studied. Pre-implantation evaluation included comprehensive genetic testing for genetic diagnosis. A novel score of genetic variants affecting genes with functional effects in the spiral ganglion was calculated. A Z-scored average of up to three post-operative speech perception tests (CNC, HINT, and AzBio) was used to assess outcome. RESULTS: Genetically determined spiral ganglion health affects cochlear implant outcomes, and when considered in conjunction with clinically determined etiology of deafness, accounts for 18.3% of the variance in postoperative speech recognition outcomes. Cochlear implant recipients with deleterious genetic variants that affect the cochlear sensory organ perform significantly better on tests of speech perception than recipients with deleterious genetic variants that affect the spiral ganglion. CONCLUSION: Etiological diagnosis of deafness including genetic testing is the single largest predictor of postoperative speech outcomes in adult cochlear implant recipients. A detailed understanding of the genetic underpinning of hearing loss will better inform pre-implant counseling. The method presented here should serve as a guide for further research into the molecular physiology of the peripheral auditory system and cochlear implants.

Noise Stress Induces an Epidermal Growth Factor Receptor/Xeroderma Pigmentosum-A Response in the Auditory Nerve.

In response to toxic stressors, cancer cells defend themselves by mobilizing one or more epidermal growth factor receptor (EGFR) cascades that employ xeroderma pigmentosum-A (XPA) to repair damaged genes. Recent experiments discovered that neurons within the auditory nerve exhibit basal levels of EGFR+XPA co-expression. This finding implied that auditory neurons in particular or neurons in general have the capacity to mobilize an EGFR+XPA defense. Therefore, the current study tested the hypothesis that noise stress would alter the expression pattern of EGFR/XPA within the auditory nerve. Design-based stereology was used to quantify the proportion of neurons that expressed EGFR, XPA, and EGFR+XPA with and without noise stress. The results revealed an intricate neuronal response that is suggestive of alterations to both co-expression and individual expression of EGFR and XPA. In both the apical and middle cochlear coils, the noise stress depleted EGFR+XPA expression. Furthermore, there was a reduction in the proportion of neurons that expressed XPA-alone in the middle coils. However, the noise stress caused a significant increase in the proportion of neurons that expressed EGFR-alone in the middle coils. The basal cochlear coils failed to mobilize a significant response to the noise stress. These results suggest that EGFR and XPA might be part of the molecular defense repertoire of the auditory nerve.

Biofunctionalized peptide-based hydrogels provide permissive scaffolds to attract neurite outgrowth from spiral ganglion neurons.

Cochlear implants (CI) allow for hearing rehabilitation in patients with sensorineural hearing loss or deafness. Restricted CI performance results from the spatial gap between spiral ganglion neurons and the CI, causing current spread that limits spatially restricted stimulation and impairs frequency resolution. This may be substantially improved by guiding peripheral processes of spiral ganglion neurons towards and onto the CI electrode contacts. An injectable, peptide-based hydrogel was developed which may provide a permissive scaffold to facilitate neurite growth towards the CI. To test hydrogel capacity to attract spiral ganglion neurites, neurite outgrowth was quantified in an in vitro model using a custom-designed hydrogel scaffold and PuraMatrix®. Neurite attachment to native hydrogels is poor, but significantly improved by incorporation of brain-derived neurotrophic factor (BDNF), covalent coupling of the bioactive laminin epitope IKVAV and the incorporation a full length laminin to hydrogel scaffolds. Incorporation of full length laminin protein into a novel custom-designed biofunctionalized hydrogel (IKVAV-GGG-SIINFEKL) allows for neurite outgrowth into the hydrogel scaffold. The study demonstrates that peptide-based hydrogels can be specifically biofunctionalized to provide a permissive scaffold to attract neurite outgrowth from spiral ganglion neurons. Such biomaterials appear suitable to bridge the spatial gap between neurons and the CI.

Regenerative efficacy of mesenchymal stromal cells from human placenta in sensorineural hearing loss.

INTRODUCTION: Hearing loss is a common chronic disorder characterized by decline of auditory function. The global population have suffered from deafness and the transplantation of stem cells is regarded as a therapeutic strategy for this disease. METHODS: We collected placenta from a total of 13 samples of full term pregnant women and isolated MSCs derived from human placenta and transplanted MSCs on deaf animal model. The normal group and the sensorineural hearing loss (SNHL) group and the experimental (transplanted MSCs) group were compared and estimated hearing level using auditory brainstem response (ABR) recordings and the otoacoustic emission (OAE) test. RESULTS: ABR threshold value and DPOAE level showed that MSCs transplantation groups was improved than the SNHL group. And the number of spiral ganglion neurons were increased in all turn of the cochlea. And there was no evidence of acute immunological rejection and inflammation response was not observed. DISCUSSION: This study is to evaluate regenerative efficacy of hearing loss by transplanting mesenchymal stromal cells (MSCs) derived from human placenta (amnion and chorion) in deaf animal model. We identified that MSCs transplantation restored auditory impairment and promoted cell regeneration. We hope to overcome sensorineural hearing loss by transplanting stem cells such as mesenchymal stromal cells (MSCs) from easily accessible adult stem cell source in placenta.

Cochlear Spiral Ganglion Neuron Apoptosis in Neonatal Mice with Murine Cytomegalovirus-Induced Sensorineural Hearing Loss.

BACKGROUND: Sensorineural hearing loss (SNHL) is the most common consequence of congenital cytomegalovirus (CMV) infection, and could result in neurological abnormalities and intellectual and developmental disabilities. PURPOSE: To explore the mechanism of murine CMV (MCMV)-induced SNHL in neonatal mice model. RESEARCH DESIGN: A repeated measures design was used. STUDY SAMPLE: Total 72 neonatal BALB/C mice (36 males and 36 females) were randomly divided into two groups. DATA COLLECTION AND ANALYSIS: MCMV suspension (50% tissue culture infective dose = 10(4.15) IU/0.1 ml, 15 µl) or physiological saline was intracranially injected into neonatal mice in the experimental or control group, respectively. Auditory brainstem response (ABR) was measured at three weeks postinjection. At 1, 3, 5, 7, 14, and 21 days postinjection, MCMV-DNA polymerase chain reaction analysis was performed to detect MCMV infection in cochlea, followed by terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end labeling analysis and immunohistochemistry staining. RESULTS: Extended latency, decreased amplitude, and increased threshold of ABR wave I were observed in the experimental group. Polymerase chain reaction test was positive from 3 to 21 days postinjection in the experimental group and negative at each time point in the control group. The average apoptosis index was higher in the experimental group than that in the control group from 3 to 21 days postinjection (p < 0.01). In addition, compared with the control group, B-cell lymphoma 2 and B-cell lymphoma 2-associated protein ratio was decreased in the experimental group (p < 0.01). CONCLUSION: Spiral ganglion neuron apoptosis was an important component of the mechanism of SNHL in MCMV-infected mice.

Fractalkine Signaling Regulates Macrophage Recruitment into the Cochlea and Promotes the Survival of Spiral Ganglion Neurons after Selective Hair Cell Lesion.

Macrophages are recruited into the cochlea in response to injury caused by acoustic trauma or ototoxicity, but the nature of the interaction between macrophages and the sensory structures of the inner ear remains unclear. The present study examined the role of fractalkine signaling in regulating the injury-evoked behavior of macrophages following the selective ablation of cochlear hair cells. We used a novel transgenic mouse model in which the human diphtheria toxin receptor (huDTR) is selectively expressed under the control of Pou4f3, a hair cell-specific transcription factor. Administration of diphtheria toxin (DT) to these mice resulted in nearly complete ablation of cochlear hair cells, with no evident pathology among supporting cells, spiral ganglion neurons, or cells of the cochlear lateral wall. Hair cell death led to an increase in macrophages associated with the sensory epithelium of the cochlea. Their numbers peaked at 14 days after DT and then declined at later survival times. Increased macrophages were also observed within the spiral ganglion, but their numbers remained elevated for (at least) 56 d after DT. To investigate the role of fractalkine signaling in macrophage recruitment, we crossed huDTR mice to a mouse line that lacks expression of the fractalkine receptor (CX3CR1). Disruption of fractalkine signaling reduced macrophage recruitment into both the sensory epithelium and spiral ganglion and also resulted in diminished survival of spiral ganglion neurons after hair cell death. Our results suggest a fractalkine-mediated interaction between macrophages and the neurons of the cochlea. SIGNIFICANCE STATEMENT: It is known that damage to the inner ear leads to recruitment of inflammatory cells (macrophages), but the chemical signals that initiate this recruitment and the functions of macrophages in the damaged ear are unclear. Here we show that fractalkine signaling regulates macrophage recruitment into the cochlea and also promotes the survival of cochlear afferents after selective hair cell lesion. Because these afferent neurons carry sound information from the cochlea to the auditory brainstem, their survival is a key determinant of the success of cochlear prosthetics. Our data suggest that fractalkine signaling in the cochlea is neuroprotective, and reveal a previously uncharacterized interaction between cells of the cochlea and the innate immune system.

Insertion trauma and recovery of function after cochlear implantation: Evidence from objective functional measures.

Partial loss and subsequent recovery of cochlear implant function in the first few weeks following cochlear implant surgery has been observed in previous studies using psychophysical detection thresholds. In the current study, we explored this putative manifestation of insertion trauma using objective functional measures: electrically-evoked compound action potential (ECAP) amplitude-growth functions (ECAP amplitude as a function of stimulus level). In guinea pigs implanted in a hearing ear with good post-implant hearing and good spiral ganglion neuron (SGN) survival, consistent patterns of ECAP functions were observed. The slopes of ECAP growth functions were moderately steep on the day of implant insertion, decreased to low levels over the first few days after implantation and then increased slowly over several weeks to reach a relatively stable level. In parallel, ECAP thresholds increased over time after implantation and then recovered, although more quickly, to a relatively stable low level as did thresholds for eliciting a facial twitch. Similar results were obtained in animals deafened but treated with an adenovirus with a neurotrophin gene insert that resulted in good SGN preservation. In contrast, in animals implanted in deaf ears that had relatively poor SGN survival, ECAP slopes reached low levels within a few days after implantation and remained low. These results are consistent with the idea that steep ECAP growth functions require a healthy population of auditory nerve fibers and that cochlear implant insertion trauma can temporarily impair the function of a healthy SGN population.

Sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation will increase in lipopolysaccharide-induced inflammation in vitro model.

BACKGROUND: With the increasing popularity of mobile phones, the potential hazards of radiofrequency electromagnetic radiation (RF-EMR) on the auditory system remain unclear. Apart from RF-EMR, humans are also exposed to various physical and chemical factors. We established a lipopolysaccharide (LPS)-induced inflammation in vitro model to investigate whether the possible sensitivity of spiral ganglion neurons to damage caused by mobile phone electromagnetic radiation (at specific absorption rates: 2, 4 W/kg) will increase. METHODS: Spiral ganglion neurons (SGN) were obtained from neonatal (1- to 3-day-old) Sprague Dawley® (SD) rats. After the SGN were treated with different concentrations (0, 20, 40, 50, 100, 200, and 400 µg/ml) of LPS, the Cell Counting Kit-8 (CCK-8) and alkaline comet assay were used to quantify cellular activity and DNA damage, respectively. The SGN were treated with the moderate LPS concentrations before RF-EMR exposure. After 24 h intermittent exposure at an absorption rate of 2 and 4 W/kg, DNA damage was examined by alkaline comet assay, ultrastructure changes were detected by transmission electron microscopy, and expression of the autophagy markers LC3-II and Beclin1 were examined by immunofluorescence and confocal laser scanning microscopy. Reactive oxygen species (ROS) production was quantified by the dichlorofluorescin-diacetate assay. RESULTS: LPS (100 µg/ml) induced DNA damage and suppressed cellular activity (P < 0.05). LPS (40 µg/ml) did not exhibit cellular activity changes or DNA damage (P > 0.05); therefore, 40 µg/ml was used to pretreat the concentration before exposure to RF-EMR. RF-EMR could not directly induce DNA damage. However, the 4 W/kg combined with LPS (40 µg/ml) group showed mitochondria vacuoles, karyopyknosis, presence of lysosomes and autophagosome, and increasing expression of LC3-II and Beclin1. The ROS values significantly increased in the 4 W/kg exposure, 4 W/kg combined with LPS (40 µg/ml) exposure, and H2O2 groups (P < 0.05, 0.01). CONCLUSIONS: Short-term exposure to radiofrequency electromagnetic radiation could not directly induce DNA damage in normal spiral ganglion neurons, but it could cause the changes of cellular ultrastructure at special SAR 4.0 W/kg when cells are in fragile or micro-damaged condition. It seems that the sensitivity of SGN to damage caused by mobile phone electromagnetic radiation will increase in a lipopolysaccharide-induced inflammation in vitro model.

[Study on the induced differentiation of induced pluripotent stem cells into cochlear hair cell-like cells and spiral ganglion neuron-like cells in vitro].

OBJECTIVE: In this study, we investigated the potential of mouse induced pluripotent stem cells (iPSC) for use as a source of transplants for the restoration of auditory hair cells and spiral ganglion neurons. METHODS: We co-cultured the mouse iPSC with the cells of the cochlear organ of Corti or the modiolus in vitro. The cochlear organ of Corti (which contains cochlear hair cells) and the modiolus (which contains auditory spiral ganglion neurons) were obtained from postnatal day 3 (P3) CD-1 ICR mice. After 18 days of coculture with the cells of newborn mouse cochleae. The expressions of hair cell markers (Myosin VIIa, Math1, Calretinin, Espin) and Spiral ganglion neuron markers [Nestin, Neurofilament-M, ß-III Tubulin, Vesicular glutamate transporter 1(VGluT1)] were detected by immunocytochemical analysis. RESULTS: Immunocytochemical analysis results indicated that the differentiated iPSC expressed auditory hair cell markers (MyosinVIIa,Math1, Calretinin, Espin ) and spiral ganglion markers (Nestin, Neurofilament-M,ß-III Tubulin,VGluT1). CONCLUSION: Mouse iPSC in virto cultured could successfully be induced to differentiate into hair cell-like cells and spiral ganglion-like cells with hair cell and spiral ganglion molecular markers.