Translocation of telomerase reverse transcriptase coincided with ATP release in postnatal cochlear supporting cells

The spontaneous bursts of electrical activity in the developing auditory system are derived from the periodic release of adenosine triphosphate(ATP)by supporting cells in the Kölliker’s organ.However,the mechanisms responsible for initiating spontaneous ATP release have not been determined.Our previous study revealed that telomerase reverse transcriptase(TERT)is expressed in the basilar membrane during the first postnatal week.Its role in cochlear development remains unclear.In this study,we investigated the expression and role of TERT in postnatal cochlea supporting cells.Our results revealed that in postnatal cochlear Kölliker’s organ supporting cells,TERT shifts from the nucleus into the cytoplasm over time.We found that the TERT translocation tendency in postnatal cochlear supporting cells in vitro coincided with that observed in vivo.Further analysis showed that TERT in the cytoplasm was mainly located in mitochondria in the absence of oxidative stress or apoptosis,suggesting that TERT in mitochondria plays roles other than antioxidant or anti-apoptotic functions.We observed increased ATP synthesis,release and activation of purine signaling systems in supporting cells during the first 10 postnatal days.The phenomenon that TERT translocation coincided with changes in ATP synthesis,release and activation of the purine signaling system in postnatal cochlear supporting cells suggested that TERT may be involved in regulating ATP release and activation of the purine signaling system.Our study provides a new research direction for exploring the spontaneous electrical activity of the cochlea during the early postnatal period.

Comparing accuracy of cochlear measurements on magnetic resonance imaging and computed tomography:A step towards radiation-free cochlear implantation

Objective:Computed Tomography(CT)and Magnetic Resonance Imaging(MRI)are commonly employed in pre-operative evaluation for cochlear implant surgery.However,with a decrease in the age of implantation,even minor radiation exposure can cause detrimental effects on children over their lifetime.The current study compares different cochlear measurements from CT and MRI scans and evaluates the feasibility of using only an MRI scan for radiological evaluation before cochlear implantation.Methods:A longitudinal observational study was conducted on 94 ears/47 children,employing CT and MRI scans.The CT and MRI scan measurements include,A value,B value,Cochlear duct length(CDL),twoturn cochlear length,alpha and beta angles to look for cochlear orientation.Cochlear nerve diameter was measured using MRI.The values were compared.Results:The mean difference between measurements from CT and MRI scans for A value,B value,CDL,and two-turn cochlear length values was 0.567±0.413 mm,0.406±0.368 mm,2.365±1.675 mm,and 2.063±1.477 mm respectively without any significant difference.The alpha and beta angle measures were comparable,with no statistically significant difference.Conclusion:The study suggests that MRI scans can be the only radiological investigation needed with no radiation risk and reduces the cost of cochlear implant program in the paediatric population.There is no significant difference between the measurements obtained from CT and MRI scans.However,observed discrepancies in cochlear measurements across different populations require regionally or race-specific standardized values to ensure accurate diagnosis and precision in cochlear implant surgery.This aspect must be addressed to ensure positive outcomes for patients.

Electrically evoked auditory nerve responses in the cochlea with normal outer hair cells

As hybrid cochlear implant devices are increasingly used for restoring hearing in patients with residual hearing, it is important to understand electrically evoked responses in cochleae having functional hair cells. To test the hypothesis that extracochlear electrical stimulation (EES) from sinusoidal current can provoke an auditory nerve response with normal frequency selectivity, the EES-evoked compound action potential(ECAP) was investigated in this study. Brief sinusoidal electrical currents, delivered via a round window electrode, were used to evoke ECAP. The ECAP waveform was observed to be the same as the acoustically evoked CAP(ACAP), except for a shorter latency. The input/output and intensity/latency functions of ACAPs and ECAPs were also similar. The maximum acoustic masking for both ACAP and ECAP occurred near probe frequencies. Since the masked tuning curve of a CAP reflects the frequency selectivity of neural excitation, these data demonstrate a highly specific activation of the auditory nerve, which would result in high degree of frequency selectivity. This frequency selectivity likely results from the cochlear traveling wave caused by electrically stimulated outer hair cells.

EFFECTS OF DIABETES ON HEARING AND COCHLEAR STRUCTURES

Diabetes mellitus (DM) is a chronic systemic disease characterized by hyperglycemia, with various patho-genic mechanisms. From absolute or relative insulin deficiency, patients with DM often demonstrate vari-ous levels of metabolic disorders. Major clinical manifestations of DM include metabolic disorders, vascu-lar lesions, circulatory disturbances and neurologic complications. Along with advances in DM research, re-ports of DM related tinnitus and hearing impairment have increased continuously. Research on DM related auditory system dysfunction has focused on cochlear microcirculation, cellular homeostasis, genetics and ag-ing. Cochlear microcirculation plays an important role in cochlear physiology and its disorders are associat-ed with many inner ear diseases. Ischemia and subsequent reperfusion seen in cochlear microcirculation dis-orders are important factors in hearing damage. Understanding cochlear microcirculation and structural as well as functional changes in DM patients with hearing loss and their causal factors will help reveal patho-genic mechanisms in diabetic hearing loss and provide new ideas in developing interventions and preventing damages caused by diabetes.

THE COCHlEAR RIBBON SYNAPTIC RESPONSE TO AMINOGIYCOSIDE OTOTOXICITY IN C57BL/6J MICE

Objective To investigate the early change of cochlear ribbon synapses on inner hair cells in response to aminoglycoside ototoxicity. Methods C57BL/6J mice received intraperitoneal injection of gentamicin (100 mg/kg/day), and the apical coil organ of Corti was examined on the 4th, 7th and 10th day (n=10). Litter-mates without gentamicin treatment served as controls (n=10). RIBEYE on the presynaptic membrane and AMPA receptors on the postsynaptic membrane were labeled with CtBP2 or GluR2/3 respectively. Three di-mension reconstruction was conducted using the 3DS MAX 8.0 software. Results There were no disruptions of outer or inner hair cells in all groups. However, the number of ribbon synapses on cochlear inner hair cells increased significantly within 7 days after gentamicin exposure (P<0.01), followed by a significant de-crease after 7 days.Conclusion During the early stage of aminoglycoside ototoxicity, increased population of cochlear ribbon synapses may indicate a significant down-regulation of synaptic function.

Lentivirus carrying the Atoh1 gene infects normal rat cochlea

Lentivirus carrying the Atohl gene can infect Corti＇s organ and express a hair-like cell surface marker in the supporting cell area. However, expression of the gene carried by adenovirus is instantaneous, which undoubtedly limits its clinical application. Lentivirus acts as a carrier that can stably and continuously express genes. In this study, the cochlear structure and hearing level were not affected, and Atohl gene carried by lentivirus promoted the production of hair-like cells in the cochlear supporting cell area. This led to expression of the hair-like cell surface marker myosin 7a 30 days after lentivirus carrying Atohl was microinjected into the cochlear round window of rats.

Detection of apoptosis by RT-PCR array in mefloquine-induced cochlear damage

Objective To investigate the occurrence and possible mechanisms of apoptosis in cochlear epithelium and spiral ganglion neurons after mefloquine treatment. Methods We used quantitative RT-PCR apoptosis-focused gene arrays （96-well, 84 apoptosis related genes） to assess changes of gene expression in the cochlear basilar membrane （hair cells-supporting cells） and spiral ganglion neurons of rat cochlear organotypic cultures treated with 100 IxM mefloquine for 3 h. Results Significant up-or down-regulation in gene expression was detected in 23 genes in the cochlear basilar membrane, and in 32 genes in the spiral ganglion neurons compared with time-matched controls. The responding genes could be classified as pro-or anti-apoptotic, and were mainly implicated in the Bcl-2, Caspase, Card, IAP, TNF ligand / TNF receptor, Death domain / Death effector domain, DNA damage / p53, and NF-kappa B families. Synthetic analysis suggested that these families could be revised to two major pathways mainly involved in t]he death receptor-mediated signaling pathway and apoptotic mitochondrial pathway. In addition, it was found that numerous anti-apoptotic genes such as Bcl2al, Birclb, Birc3, Birc4, Bnipl, Cflar, II10, Lhx4, Mcll, Nfkbl, Prlr, Prok2, and TNF were greatly up-regulated in the cochlear tissue, which might imply the co-existence of protective response in the ceils at the early stage of mefloquine-induced damage.

Notch pathway inhibitor DAPT enhances Atoh1 activity to generate new hair cells in situ in rat cochleae

Atoh1 overexpression in cochlear epithelium induces new hair cell formation. Use of adenovirus-mediated Atoh1 overexpression has mainly focused on the rat lesser epithelial ridge and induces ectopic hair cell regeneration. The sensory region of rat cochlea is difficult to transfect, thus new hair cells are rarely produced in situ in rat cochlear explants. After culturing rat cochleae in medium containing 10% fetal bovine serum, adenovirus successfully infected the sensory region as the width of the supporting cell area was significantly increased. Adenovirus encoding Atoh1 infected the sensory region and induced hair cell formation in situ. Combined application of the Notch inhibitor DAPT and Atoh1 increased the Atoh1 expression level and decreased hes1 and hes5 levels, further promoting hair cell generation. Our results demonstrate that DAPT enhances Atoh1 activity to promote hair cell regeneration in rat cochlear sensory epithelium in vitro.

Ligustrazine facilitates hair cell regeneration in the cochlea following gentamicin ototoxicity

BACKGROUND： Ligustrazine （tetramethylpyrazine） decreases ototoxicity induced by gentamicin and facilitates hair cell regeneration and repair, but the precise mechanisms remain controversial.OBJECTIVE： To explore the protective effects of ligustrazine on gentamicin ototoxicity by determining heat shock protein 70 mRNA and protein expression in the cochlear stria vascularis of guinea pigs at different time points.DESIGN, TIME AND SETTING： The randomized, controlled study was performed at the Laboratory of Physiology, Shenyang Medical College of China in 2007.MATERIALS： Ligustrazine parenteral solution （Qiqihar Pharmaceutical Factory, China） and gentamicin sulfate （Shenyang First Pharmaceutical Factory, China） were used in this experiment.METHODS： White guinea pigs with red eyes were randomly intraperitoneally administered gentamicin sulfate injection ＋ saline, gentamicin sulfate injection ＋ ligustrazine, and ligustrazine ＋ saline, respectively.MAIN OUTCOME MEASURES： Auditory brains tern response threshold was measured. Immunohistochemistry, in situ hybridization, and image analyzing techniques were utilized to determine heat shock protein 70 mRNA and protein expression in cochlear stria vascularis of guinea pigs.RESULTS： Following gentamicin ototoxicity, the auditory brainstem response threshold increased, peaked on day 3, and then decreased with increased time after drug withdrawal. The auditory brainstem response threshold was significantly diminished following ligustrazine intervention, but recovered to normal on day 30 （P〉0.05）. Heat shock protein 70 expression also increased, peaked on day 3, and then decreased in the cochlear stria vascularis of guinea pigs following gentamicin ototoxicity. Ligustrazine intervention resulted in decreased heat shock protein 70 expression in the cochlear stria vascularis, which recovered to normal on day 14. Heat shock protein 70 mRNA expression increased in the cochlear stria vascularis following gentamicin ototoxicity, but ligustrazine intervention resulted in decreased levels. CONCLUSION： Ligustrazine significantly ameliorated gentamicin ototoxicity by reducing heat shock protein 70 mRNA and protein expression in the cochlear stria vascularis.

Migration of R28 Retinal Precursor Cells into Cochlear and Vestibular Organs

Damaged hair cells and neurons in the inner ear generally can not be replaced in mammals. The loss of these cells causes permanent functional disorders in both the cochlear and vestibular systems. Transplantation of retinal precursor cells, R28 cells, into inner ear tissue may help replace missing cells. The aim of the current project was to induce R28 cell transdifferentiation into cochlear and vestibular cell types under culture conditions. The first part was related to R28 cell labeling with DiI fluorescence that would help identify and track R28 cells. The second part involved co-culturing R28 cells in cochlear and vestibular organotropic cultures or isolated spiral ganglion neurons. The results suggest that R28 cells have the potential to differentiate into supporting cell types and spiral ganglion neurons in serum free medium, probably under the influence of diffusible signals from inner ear tissues. This information is useful for future efforts in inducing stem cell differentiation in the inner ear to replace lost sensory and neural cells.

Antioxidative stress-induced damage in cochlear explants

The imbalance of reactive oxygen species and antioxidants is considered to be an important factor in the cellular injury of the inner ear. At present, great attention has been placed on oxidative stress. However,little is known about fighting oxidative stress. In the current study, we evaluated antioxidant-induced cochlear damage by applying several different additional antioxidants. To determine whether excessive antioxidants can cause damage to cochlear cells, we treated cochlear explants with 50 m M M40403, a superoxide dismutase mimetic, 50 m M coenzyme Q-10, a vitamin-like antioxidant, or 50 m M d-methionine, an essential amino acid and the important antioxidant glutathione for 48 h. Control cochlear explants without the antioxidant treatment maintained their normal structures after incubation in the standard serum-free medium for 48 h, indicating the maintenance of the inherent oxidative and antioxidant balance in these cochlear explants. In contrast, M40403 and coenzyme Q-10-treated cochlear explants displayed significant hair cell damage together with slight damage to the auditory nerve fibers.Moreover, d-methiodine-treated explants exhibited severe damage to the surface structure of hair cells and the complete loss of the spiral ganglion neurons and their peripheral fibers. These results indicate that excessive antioxidants are detrimental to cochlear cells, suggesting that inappropriate dosages of antioxidant treatments can interrupt the balance of the inherent oxidative and antioxidant capacity in the cell.

Super-resolution immunohistochemistry study on CD4 and CD8 cells and the relation to macrophages in human cochlea

Recently, the human cochlea has been shown to contain numerous resident macrophages under steady-state. The macrophages accumulate in the stria vascularis, among the auditory nerves, and are also spotted in the human organ of Corti. These macrophages may process antigens reaching the cochlea by invasion of pathogens and insertion of CI electrode. Thus, macrophages execute an innate, and possibly an adaptive immunity. Here, we describe the molecular markers CD4 and CD8 of T cells, macrophage markers MHCⅡ and CD11b, as well as the microglial markers TEME119 and P2Y12, in the human cochlea. Immunohistochemistry and the advantageous super-resolution structured illumination microscopy(SR-SIM) were used in the study. CD4^+ and CD8^+ cells were found in the human cochleae. They were seen in the modiolus in a substantial number adjacent to the vessels, in the peripheral region of the Rosenthal's canal, and occasionally in the spiral ligament. While there are a surprisingly large number of macrophages in the stria vascularis as well as between the auditory neurons,CD4^+ and CD8^+ cells are hardly seen in these areas, and neither are seen in the organ of Corti. In the modiolus,macrophages, CD4^+ and CD8^+ cells appeared often in clusters. Interaction between these different cells was easily observed with SR-SIM, showing closely placed cell bodies, and the processes from macrophages reaching out and touching the lymphocytes. Otherwise the CD4^+ and CD8^+ cells in human cochlear tissue are discretely scattered. The possible roles of these immune cells are speculated.

Spontaneous Proliferation in Organotypic Cultures of Mouse Cochleae

Cells in mammalian cochleae virtually stop proliferation and exit cellular circle before birth. Consequently, hair cells and spiral ganglion neurons destroyed by ototoxic factors cannot be replaced through proliferative regeneration. However, substantial proliferation occurs in organotypic cultures of cochleae from postnatal mice. In the present study, we studied the time course of proliferative growth in cultures of mouse cochlea explants obtained from up to 12 postnatal days. The mitotic nature of this growth was confirmed by bromodeoxyuridine (BrdU) staining and expression of proliferation cell nuclear antigen (PCNA) evaluated with real-time quantitative poly-merase chain reaction(RT-PCR). Similar growth time course was found in the cochlear explants of different postnatal ages. The new growth reached its maximum at around 2 days in culture followed by a slow-down, and virtually stopped after 5 days of culture. The possible mechanisms and the significance of this proliferation are discussed.

Effect of Hypertension on Hearing Function,LDH and ChE of the Cochlea in Older Rats

The relationship between the hypertension and the aging process of hearing organ was investigated Twenty Wistar 3-month old rats and 20 Wistar 12-month old rats, 20 spontaneously hypertensive rat stroke-prone (SHRSP) 3-month old rats and 20 SHRSP 12-month old rats free of middle ear infections as observed under otomicroscopy, with normal tympanic membrane and auricle reflex, were selected to be divided into two experimental groups and two control groups respectively The tail artery blood pressure was measured non-invasively The threshold of auditory brainstem response (ABR) was measured by Spirit TM evoked potential meter The LDH and ChE staining in the inner ear was performed and the optical density was analyzed by the HPIAS analysis system The results showed that there was no difference in the ABR thresholds, the activities of LDH and ChE between Wistar 3-month old group and SHRSP 3-month old group ( P >0 05) The mean value of ABR threshold and the activities of LDH and ChE in the Wistar 12-month old group at relevant sections were significantly greater than those in the two 3-month old groups ( P< 0 05), whereas the mean value of ABR threshold and the activities of LDH and ChE in the SHRSP 12-month old group at relevant sections were significantly higher than those in the 3-month old control group ( P< 0 01) It was concluded that presbycusis existed in the Wistar 12-month old group rats The glycogenosis and the abnormal secretion of neural transmitter were discerned after hypertension All the above factors may worsen the aging of the hearing system

The Expression of VIP and SP in the Cochlea of Spontaneously Hypertensive Rats and Its Implication

To investigate the expression of vasoactive intestinal peptide (VIP) and substance P (SP) in the cochlea of spontaneously hypertensive rat (SHR), and to assess the function of VIP and SP in the cochlea following the damage of hypertension, hearing thresholds of ABR were observed and the fixative (4% paraformaldehyde) was pumped through the circulatory system. Adult Wistar rats (3 months, n=20) served as the control group and SHRs (3 months, n=20) as the hypertension group. Bullas were taken out and cochleas were irrigated in vitro with the same fixative. The number of base turn's spiral ganglions in the sections was counted. The expression of VIP and SP were detected by SABC method and the images of the sections were analyzed. The number of base turn's spiral ganglsons in the hypertension group was significantly less than in the normal group (P<0.01). VIP and SP were expressed in the spiral ganglion cytoplasma and stria vascularis of the two groups. There were no significant difference in the expression of VIP and SP in spiral ganglion cytoplasma (P>0.05) between the two groups. However, in stria vascularis the expression of VIP in the hypertension group was higher than in the normal group (P<0.05), and no significant difference in SP was found between the two groups. It was suggested that VIP not only contributed to the regulation of the cochlea microcirculation, but also made the neurotransmitter in the pathway of the auditory system. However, SP made only the neurotransmitter in the pathway of the auditory system.

Cell junction proteins within the cochlea: A review of recent research

Cell-cell junctions in the cochlea are highly complex and well organized. The role of these junctions is to maintain structural and functional integrity of the cochlea. In this review, we describe classification of cell junction-associated proteins identified within the cochlea and provide a brief overview of the function of these proteins in adherent junctions, gap junctions and tight junctions. Copyright ? 2016, PLA General Hospital Department of Otolaryngology Head and Neck Surgery. Production and hosting by Elsevier (Singapore) Pte Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Lead neurotoxicity in rat cochlear organotypic cultures

Lead is a major environmental toxicant throughout the world.Lead can induce severe neurotoxicity including irreversible hearing impairment.Many in vivo studies have shown that lead damages the auditory nervous system,but has little or no effect on cochlear sensory hair cells.To gain insights on lead ototoxic and neurotoxic effects in vitro,lead acetate (LA) was applied to postnatal day 3-4 rat cochlear organotypic cultures for 24 or 72 h with doses of 0.1,0.5,1,2 or 4 mM.After 24 or 72 h treatment with lead acetate,nearly all of cochlear sensory hair cells were intact.However,after 72 h treatment,the peripheral auditory nerve fibers projecting to the hair cells and the spiral ganglion neurons (SGN) were damaged when lead concentration exceeded 2 mM.Our results indicated that 72 h treatment with only the high doses (> 2 mM) of lead actate damaged SGNs and peripheral nerve fibers;hair cells remained structurally intact even after 4 mM treatment.These results show that lead primarily damages cochlear nerve fibers andSGNratherthanhaircells.

The Effect of Nitrous Oxide and Isoflurane on the Total RNA Yield from the Cochlea of the Rats

The possible mechanism of inhalation anesthetics on the internal auditory impairment of the rat was investigated by determining the effect of nitrous oxide （N2O） and isoflurane on the total RNA yield from the cochlea of the rats. Thirty healthy Wistar rats were randomly divided into 3 groups： group C （control group, n=10） with a 3-h unremitting inhalation of 50% 02, group N （experiment group, n= 10） with a continuous inhalation of 50% N20＋50% O2for 3 h, and group I （experiment group, n=10） with a 3-h sustained inhalation of 2.5% isoflurane. The TRIzol in combination with RNeasy was used to respectively extract the total RNA from cochlea of rats in the 3 groups. Spectrophotometry was used to detect total RNA yield and electrophoresis to detect the quality. The total RNA extracted from the cochlea of the rats in the groups C and N was 7.69 and 6.51 μg, respectively. There was a 15% decrease in the N group as compared with group C. The total RNA from the rats in the group I was 7.32μg, and there was hardly any change in the group as compared with the group C. The value of A260/A280 in groups C, N and I was 2.07, 2.04 and 2.04, respectively, showing a very high RNA purity. The result of gel electrophoresis suggested that there was no degradation in the total RNA. It was suggested that the interference of N20 on the cochlear RNA yield might be one of the reasons which cause an injury of the ear. The isoflurane shows no harm on the hearing.

HIGH-THROUGHPUT TECHNOLOGIES FOR GENE EXPRESSION ANALYSES:WHAT WE HAVE LEARNED FOR NOISE-INDUCED COCHLEAR DEGENERATION

Noise-induced hearing loss is a common cause of acquired hearing loss in the adult population. Acoustic overstimulation causes cochlear damage through mechanical stress to the tissue. Consequently, complex molec-ular changes are initiated, and these changes lead to morphological and biological alterations in the cochlea, which in turn compromise the cochlear function and cause hearing loss. In the past 10 years, there have been significant advances in our understanding of the molecular mechanisms of noise-induced hearing loss. These advances are attributed, in part, to the development of high-throughput technologies for the global analyses of molecular changes. In this review, we briefly describe the newly developed methods for investigating the mo-lecular responses of the cochlea to acoustic trauma and the knowledge generated from these studies. We also discuss the strengths and limitations of each technique and the major challenges to investigate cochlear degen-eration following acoustic injury.

Downregulation of Inwardly Rectifying Potassium Channel 5.1 Expression in C57BL/6J Cochlear Lateral Wall

Age-related hearing loss（AHL） is one of the most common sensory disorders among elderly persons. The inwardly rectifying potassium channel 5.1（Kir5.1） plays a vital role in regulating cochlear K~＋ circulation which is necessary for normal hearing. The distribution of Kir5.1 in C57BL/6J mice cochleae, and the relationship between the expression of Kir5.1 and the etiology of AHL were investigated. Forty C57BL/6J mice were randomly divided into four groups at 4, 12, 24 and 52 weeks of age respectively. The location of Kir5.1 was detected by immunofluorescence technique. The m RNA and protein expression of Kir5.1 was evaluated in mice cochleae using real-time polymerase-chain reactions（RT-PCR） and Western blotting respectively. Kir5.1 was detected in the type Ⅱ and Ⅳfibrocytes of the spiral ligament in the cochlear lateral wall of C57BL/6J mice. The expression levels of Kir5.1 m RNA and protein in the cochleae of aging C57BL/6J mice were down-regulated. It was suggested that the age-related decreased expression of Kir5.1 in the lateral wall of C57BL/6J mice was associated with hearing loss. Our results indicated that Kir5.1 may play an important role in the pathogenesis of AHL.