Correlation of PDCD5 and Apoptosis in Hair Cells and Spiral Ganglion Neurons of Different Age of C57BL/6J Mice

This study examined the expression pattern of programmed cell death 5 (PDCD5) in co-chlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice.Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3,6,9 or 12 months).PDCD5 expression was detected by using immunohistochemistry,real-time PCR and Western blot.Morphological change of the cochleae was also evaluated by using immunoassay.The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice,as well as gradually increased apoptosis of cochlear hair cells and SGNs.In addition,we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing.It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs,and thereby plays a role in the pathogenesis of presbycusis.Thus,PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.

Salicylate enhances expression and function of NMDA receptors in cochlear spiral ganglion neurons

Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons （SGNs）. Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR （FQ-PCR）. NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA （0. 1mM and 0.5 mM） applied with salicylate （5 mM）, however, induced inward currents （212.6±15.2pA, n=5; 607.9±44.3pA, n=5） in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.

Gamma-aminobutyric acid A receptor and N-methyl-D-aspartate receptor subunit expression in rat spiral ganglion neurons

BACKGROUND： Gamma-aminobutyric acid A （GABAA） and N-methyl-D-aspartate （NMDA） receptors are significant receptors in the central nervous system. An understanding of GABAA and NMDA receptor expression in spiral ganglion neurons （SGN） provides information for the functional role of these receptors in the auditory system. OBJECTIVE： To investigate mRNA expression of GABAA receptor （GABAAR） and NMDA receptor （NMDAR） subunits in the rat SGN. DESIGN, TIME AND SETTING： This in vitro, molecular biological study was performed at the Laboratory of Otolaryngology-Head and Neck Surgery, Guangxi Medical University, China from July 2007 to May 2008. MATERIALS： Reverse Transcriptase Kit and Taq DNA polymerase were purchased from Fermentas Burlington, ON, Canada; GABAAR and NMDAR primers were purchased from Shanghai Sangon, Shanghai, China. METHODS： SGN from 3-5 day postnatal Wistar rats was collected for primary cultures, mRNA expression of GABAAR and NMDAR subunits in the SGN was determined by reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURES： Expression levels of GABAAR and NMDAR subunits were determined by quantitative analysis. RESULTS： GABAAR subunits （αl 6, β1 3, and y1 3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were detected in the SGN. In α subunit genes of GABAAR, α1 and α3 expression was similar （P 〉 0.05） and greater than the other subunits. Of the β subunit genes, β1 subunit mRNA levels were greater than β2 and β3. Of the y subunit genes, y2 subunit mRNA levels were greater than y1 and y3. NR1 mRNA expression was the greatest of NMDAR subunits. CONCLUSION： GABAAR subunits （α1 6, β1-3, and y1-3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were expressed in the rat SGN. Through comparison of GABAAR and NMDAR subunit expression, possible GABAAR combinations, as well as highly expressed subunit combinations, were estimated, which provided information for pharmacological and electrophysiological characteristics of GABAAR in the auditory system.

(-)-Epigallocatechin-3-gallate protects spiral ganglion neurons against amikacin-induced apoptosis

Morphology of spiral ganglion neurons （SGNs） in Sprague-Dawley rats before and after amikacin treatment was observed by transmission electron microscopy. Amikacin induced cochlear SGN apoptosis. Immunohistochemical staining and RT-PCR revealed a decrease in Bcl-2 protein ex-pression, and an increase in Bax protein, caspase-3 protein and caspase-6 mRNA expression fol-lowing amikacin treatment. （-）-Epigallocatechin-（3）-gallate （EGCG） inhibited SGN Bax protein, caspase-3 protein and caspase-6 mRNA expression, and enhanced Bcl-2 protein expression, thereby decreasing SGN apoptosis. Results demonstrated that EGCG can protect SGNs against amikacin-induced injury.

Abnormal Innervation,Demyelination,and Degeneration of Spiral Ganglion Neurons as Well as Disruption of Heminodes are Involved in the Onset of Deafness in Cx26 Null Mice

GJB2 gene mutations are the most common causes of autosomal recessive non-syndromic hereditary deafness.For individuals suffering from severe to profound GJB2-related deafness,cochlear implants have emerged as the sole remedy for auditory improvement.Some previous studies have highlighted the crucial role of preserving cochlear neural components in achieving favorable outcomes after cochlear implantation.Thus,we generated a conditional knockout mouse model(Cx26-CKO)in which Cx26 was completely deleted in the cochlear supporting cells driven by the Sox2 promoter.The Cx26-CKO mice showed severe hearing loss and massive loss of hair cells and Deiter’s cells,which represented the extreme form of human deafness caused by GJB2 gene mutations.In addition,multiple pathological changes in the peripheral auditory nervous system were found,including abnormal innervation,demyelination,and degeneration of spiral ganglion neurons as well as disruption of heminodes in Cx26-CKO mice.These findings provide invaluable insights into the deafness mechanism and the treatment for severe deafness in Cx26-null mice.

Inhibition of the NLRP3 inflammasome attenuates spiral ganglion neuron degeneration in aminoglycoside-induced hearing loss

Aminoglycosides are a widely used class of antibacterials renowned for their effectiveness and broad antimicrobial spectrum.However,their use leads to irreversible hearing damage by causing apoptosis of hair cells as their direct target.In addition,the hearing damage caused by aminoglycosides involves damage of spiral ganglion neurons upon exposure.To investigate the mechanisms underlying spiral ganglion neuron degeneration induced by aminoglycosides,we used a C57BL/6J mouse model treated with kanamycin.We found that the mice exhibited auditory deficits following the acute loss of outer hair cells.Spiral ganglion neurons displayed hallmarks of pyroptosis and exhibited progressive degeneration over time.Transcriptomic profiling of these neurons showed significant upregulation of genes associated with inflammation and immune response,particularly those related to the NLRP3 inflammasome.Activation of the canonical pyroptotic pathway in spiral ganglion neurons was observed,accompanied by infiltration of macrophages and the release of proinflammatory cytokines.Pharmacological intervention targeting NLRP3 using Mcc950 and genetic intervention using NLRP3 knockout ameliorated spiral ganglion neuron degeneration in the injury model.These findings suggest that NLRP3 inflammasome-mediated pyroptosis plays a role in aminoglycoside-induced spiral ganglion neuron degeneration.Inhibition of this pathway may offer a potential therapeutic strategy for treating sensorineural hearing loss by reducing spiral ganglion neuron degeneration.

Recent advances in the application of MXenes for neural tissue engineering and regeneration

Transition metal carbides and nitrides(MXenes)are crystal nanomaterials with a number of surface functional groups such as fluorine,hydroxyl,and oxygen,which can be used as carriers for proteins and drugs.MXenes have excellent biocompatibility,electrical conductivity,surface hydrophilicity,mechanical properties and easy surface modification.However,at present,the stability of most MXenes needs to be improved,and more synthesis methods need to be explored.MXenes are good substrates for nerve cell regeneration and nerve reconstruction,which have broad application prospects in the repair of nervous system injury.Regarding the application of MXenes in neuroscience,mainly at the cellular level,the long-term in vivo biosafety and effects also need to be further explored.This review focuses on the progress of using MXenes in nerve regeneration over the last few years;discussing preparation of MXenes and their biocompatibility with different cells as well as the regulation by MXenes of nerve cell regeneration in two-dimensional and three-dimensional environments in vitro.MXenes have great potential in regulating the proliferation,differentiation,and maturation of nerve cells and in promoting regeneration and recovery after nerve injury.In addition,this review also presents the main challenges during optimization processes,such as the preparation of stable MXenes and long-term in vivo biosafety,and further discusses future directions in neural tissue engineering.

Basic flbroblast growth factor protects auditory neurons and hair cells from noise exposure and glutamate neurotoxicity

The purpose of the present study was to determine protectivie effects of basic fibroblast growth factor (bFGF) on cochlear neurons and hair cells in vitro and in vivo. In experiment I, cultured spiral ganglion neurons (SGNs) prepared from P3 mice were exposed to 20mM glutamate for 2 hours before the culture medium was replaced with fresh medium containing 0, 25, 50, and 100 ng/ml bFGF, respectively. Fourteen days later, all cultures were fixed with 4% paraformaldehyde, and stained with 1% toluidine blue. The number of surviving SGNs were counted and the length of SGNs neurites were measured. Exposure to 20 mM glutamate for 24 hours resulted in an inhibition on neurite outgrowth of SGNs and elevated cell death. Treatment of the cultures with bFGF led to promotion of neurite outgrowth and elevated number of surviving SGNs. Effects of bFGF were dose dependent with the highest potency at 100 ng/ml. In experiment Ⅱ, in vivo studies were carried out with guinea pigs in which bFGF or artificial perilymph was perfused into the cochlea to assess possible protective effects of bFGF on cochlear hair cells and compound action potentials(CAP). The CAPs were measured before, immediatly and 48 hours after exposure to noise. Significant differences in CAP were observed (p＜0. 05 ) among the bFGF perfused group, control group(t =3. 896 ) and artificial perilymph perfused group (t =2. 520) at 48 hours after noise exposure, Cochleae were removed and hair cell Loss was analyzed in surface preparations prepared from all experimental animals. Acoustic trauma caused loss of 651 and 687 inner hair cells in the control and artificial perilymph perfused group, respectively. In sharp contrast, only 31 inner hair cells were lost in the bFGF perfused ears. Similarly, more outer hair cells died in the control and perilymph perfuesed group (41830 and 41968, respectively) than in the group treated with bFGF (34258). Our results demonstrate that bFGF protected SGNs against glutmate neurotoxicity in vitro. In addition, treatment with bFGF also protected hair cells from acoustic trauma.

Inhibition of histone methyltransferase PRMT5 attenuates cisplatininduced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway

This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5(PRMT5)in cisplatin-induced hearing loss.The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry,apoptosis assays,and auditory brainstem response.The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction(CUT&Tag-qPCR)analyses in the HEI-OC1 cell line.Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species.CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene,thus activating the expression of Pik3ca.These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatininduced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.

Gene expression in cisplatin ototoxicity and protection with p53 inhibitor

Cisplatin damages cochlear hair cells and spiral ganglion neurons through cell death signaling pathways that are not fully understood. We used focused apoptosis gene microarrays to study early changes in gene expres- sion in cochlear cultures from P3 neonatal rats treated with cisplatin (0.2 mM). After 12 hours of cisplatin treat- ment, more than 50% of the 96 genes on the array showed a significant decrease in expression, consistent with widespread cell death. However, after 3 hours of cisplatin treatment, 10 genes showed significant increase in ex- pression in total cochlear tissue. In experiments with subsets of cochlear tissues, at 3h, cisplatin induced increased expression of 12 genes in the cochlear sensory epithelium (basilar membrane) and 11 genes in the spiral ganglion (tissue of Rosenthal’s canal, containing the spiral ganglion). These included pro- and anti-apoptotic genes in- volved in the p53 signaling pathway, TNF receptor family, NF-kappaB pathway, death domain family, death effec- tor domain family, Bcl-2 family, CARD family, TRAF family, and GTP signal transduction. Although the changes in gene expression showed an overlap between basilar membrane and spiral ganglion, other changes, which may reflect the unique response of each tissue, were also observed. Pifithrin-α blocked cisplatin-induced up-regulation of genes in the p53 signaling pathway when assayed by both superarray and real time PCR. The data add to our understanding of the involvement of p53 in cisplatin-induced ototoxicity and otoprotection, conferred by the p53 inhibitor Pifithrin-α.

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.

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.

Trimethyltin-induced cochlear degeneration in rat

Trimethyltin(TMT) is an occupational and environmental health hazard behaving as a potent neurotoxin known to affect the central nervous system as well as the peripheral auditory system.However,the mechanisms underlying TMT-induced ototoxicity are poorly understood.To elucidate the effects of TMT on the cochlea,a single injection of 4 or 8 mg/kg TMT was administered intraperitoneally to adult rats.The compound action potential(CAP) threshold was used to assess the functional status of the cochlea and histological techniques were used to assess the condition of the hair cells and auditory nerve fibers.TMT at 4 mg/kg produced a temporary CAP threshold elevation of 25-60 dB that recovered by 28 d post-treatment.Although there was no hair cell loss with the 4 mg/kg dose,there was a noticeable loss of auditory nerve fibers particularly beneath the inner hair cells.TMT at 8 mg/kg produced a large permanent CAP threshold shift that was greatest at the high frequencies.The CAP threshold shift was associated with the loss of outer hair cells and inner hair cells in the basal,high-frequency region of the cochlea,considerable loss of auditory nerve fibers and a significant loss of spiral ganglion neurons in the basal turn.Spiral ganglion neurons showed evidence of soma shrinkage and nuclear condensation and fragmentation,morphological features of apoptotic cell death.TMT-induced damage was greatest in the high-frequency,basal region of the cochlea and the nerve fibers beneath the inner hair cells were the most vulnerable structures.

Stem cell-based approaches: Possible route to hearing restoration?

Disabling hearing loss is the most common sensorineural disability worldwide.It affects around 466 million people and its incidence is expected to rise to around 900 million people by 2050,according to World Health Organization estimates.Most cases of hearing impairment are due to the degeneration of hair cells(HCs)in the cochlea,mechano-receptors that transduce incoming sound information into electrical signals that are sent to the brain.Damage to these cells is mainly caused by exposure to aminoglycoside antibiotics and to some anti-cancer drugs such as cisplatin,loud sounds,age,infections and genetic mutations.Hearing deficits may also result from damage to the spiral ganglion neurons that innervate cochlear HCs.Differently from what is observed in avian and nonmammalian species,there is no regeneration of missing sensory cell types in the adult mammalian cochlea,what makes hearing loss an irreversible process.This review summarizes the research that has been conducted with the aim of developing cell-based strategies that lead to sensory cell replacement in the adult cochlea and,ultimately,to hearing restoration.Two main lines of research are discussed,one directed toward the transplantation of exogenous replacement cells into the damaged tissue,and another that aims at reactivating the regenerative potential of putative progenitor cells in the adult inner ear.Results from some of the studies that have been conducted are presented and the advantages and drawbacks of the various approaches discussed.

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.

Glutamate receptor antagonist and neurotrophin can protect inner ear against damage

In this study,I focused on finding a mean of protecting against hearing loss.By infusing the cochlea with the neurotrophin factor,NT-3 alone or combined treatment with MK 801,a NMDA receptor antagonist I found hearing loss was attenuated and spiral ganglion neuron loss was nearly totally protected indicating that the importance of the combined treatment of NT-3 and NMDA receptor antagonists in the treatment of hearing disorders.

Availability and safety assessment of infrared neural stimulation at high repetition rate through an implantable optrode

An implantable optrode with micro-thermal detectors was designed to investigate the availability and safety of INS using high repetition rates.Optical auditory brainstem responses(oABRs)were recorded in normal-hearing guinea pigs,and the energy thresholds,pulse durations,and ampli-tudes evoked by the varied stimulus repetitions were analyzed.Stable oABRs could be evoked through INS even as the repetition rate of stimulation reached 19 kHz.The energy threshold of oABRs was elevated,the amplitudes decreased as pulse durations increased and repetition rates were higher,and the latencies were delayed as the pulse durations increased.The temperature variation curves on the site of stimulation significantly increased as the pulse duration increased to 400μs.INS elevated the temperature around the stimulus site area via thermal accumulation during radiation,especially when higher repetition stimuli were used.Our results demonstrate that high repetition infrared stimulations can safely evoke stable and available oABRs in normal-hearing guinea pigs.

Anatomy and physiology of peripheral auditory system and commen causes of hearing loss

For Otolaryngologist,it is the most important to know the principle of anatomy,physiology and common ototoxicity.Short but more concise summary has been sum up in the following review in order to help young ENT doctor to understand the importance of this basic knowledge.

Advance and Application of Single‑cell Transcriptomics in Auditory Research

Hearing loss and deafness,as a worldwide disability disease,have been troubling human beings.However,the auditory organ of the inner ear is highly heterogeneous and has a very limited number of cells,which are largely uncharacterized in depth.Recently,with the development and utilization of single-cell RNA sequencing(scRNA-seq),researchers have been able to unveil the complex and sophisticated biological mechanisms of various types of cells in the auditory organ at the single-cell level and address the challenges of cellular heterogeneity that are not resolved through by conventional bulk RNA sequencing(bulk RNAseq).Herein,we reviewed the application of scRNA-seq technology in auditory research,with the aim of providing a reference for the development of auditory organs,the pathogenesis of hearing loss,and regenerative therapy.Prospects about spatial transcriptomic scRNA-seq,single-cell based genome,and Live-seq technology will also be discussed.

Inhibiting DNA methylation alleviates cisplatininduced hearing loss by decreasing oxidative stress-induced mitochondria-dependent apoptosis via the LRP1-PI3K/AKT pathway

Cisplatin-related ototoxicity is a critical side effect of chemotherapy and can lead to irreversible hearing loss.This study aimed to assess the potential effect of the DNA methyltransferase(DNMT)inhibitor RG108 on cisplatin-induced ototoxicity.Immunohistochemistry,apoptosis assay,and auditory brainstem response(ABR)were employed to determine the impacts of RG108 on cisplatin-induced injury in murine hair cells(HCs)and spiral ganglion neurons(SGNs).Rhodamine 123 and TMRM were utilized for mitochondrial membrane potential(MMP)assessment.Reactive oxygen species(ROS)amounts were evaluated by Cellrox green and Mitosox-red probes.Mitochondrial respiratory function evaluation was performed by determining oxygen consumption rates(OCRs).The results showed that RG108 can markedly reduce cisplatin induced damage in HCs and SGNs,and alleviate apoptotic rate by protecting mitochondrial function through preventing ROS accumulation.Furthermore,RG108 upregulated BCL-2 and downregulated APAF1,BAX,and BAD in HEI-OC1 cells,and triggered the PI3K/AKT pathway.Decreased expression of low-density lipoprotein receptor-related protein 1(LRP1)and high methylation of the LRP1 promoter were observed after cisplatin treatment.RG108 treatment can increase LRP1expression and decrease LRP1 promoter methylation.In conclusion,RG108 might represent a new potential agent for preventing hearing loss induced by cisplatin via activating the LRP1-PI3K/AKT pathway.