Tissue engineering strategies for spiral ganglion neuron protection and regeneration.

Cochlear implants can directly activate the auditory system's primary sensory neurons, the spiral ganglion neurons (SGNs), via circumvention of defective cochlear hair cells. This bypass restores auditory input to the brainstem. SGN loss etiologies are complex, with limited mammalian regeneration. Protecting and revitalizing SGN is critical. Tissue engineering offers a novel therapeutic strategy, utilizing seed cells, biomolecules, and scaffold materials to create a cellular environment and regulate molecular cues. This review encapsulates the spectrum of both human and animal research, collating the factors contributing to SGN loss, the latest advancements in the utilization of exogenous stem cells for auditory nerve repair and preservation, the taxonomy and mechanism of action of standard biomolecules, and the architectural components of scaffold materials tailored for the inner ear. Furthermore, we delineate the potential and benefits of the biohybrid neural interface, an incipient technology in the realm of implantable devices. Nonetheless, tissue engineering requires refined cell selection and differentiation protocols for consistent SGN quality. In addition, strategies to improve stem cell survival, scaffold biocompatibility, and molecular cue timing are essential for biohybrid neural interface integration.

Auditory hair cells and spiral ganglion neurons regenerate synapses with refined release properties in vitro.

Ribbon synapses between inner hair cells (IHCs) and type I spiral ganglion neurons (SGNs) in the inner ear are damaged by noise trauma and with aging, causing "synaptopathy" and hearing loss. Cocultures of neonatal denervated organs of Corti and newly introduced SGNs have been developed to find strategies for improving IHC synapse regeneration, but evidence of the physiological normality of regenerated synapses is missing. This study utilizes IHC optogenetic stimulation and SGN recordings, showing that, when P3-5 denervated organs of Corti are cocultured with SGNs, newly formed IHC/SGN synapses are indeed functional, exhibiting glutamatergic excitatory postsynaptic currents. When using older organs of Corti at P10-11, synaptic activity probed by deconvolution showed more mature release properties, closer to the specialized mode of IHC synaptic transmission crucial for coding the sound signal. This functional assessment of newly formed IHC synapses developed here, provides a powerful tool for testing approaches to improve synapse regeneration.

Extrasynaptic distribution of NMDA receptors in cochlear inner hair cell afferent signaling complex.

OBJECTIVE: The distribution and role of NMDA receptors is unclear in the afferent signaling complex of the cochlea. The present study aimed to examine the distribution of NMDA receptors in cochlear afferent signaling complex of the adult mouse, and their relationship with ribbon synapses of inner hair cells (IHCs) and GABAergic efferent terminals of the lateral olivocochlear (LOC). METHODS: Immunofluorescence staining in combination with confocal microscopy was used to investigate the distribution of glutamatergic NMDA and AMPA receptors in afferent terminals of SGNs, and their relationship with ribbon synapses of IHCs and GABAergic efferent terminals of LOC. RESULTS: Terminals with AMPA receptors along with Ribbons of IHC formed afferent synapses in the basal pole of IHCs, and those with NMDA receptors were mainly distributed longitudinally in the IHCs nuclei region. Significant difference was found in the distribution of NMDA and AMPA receptors in IHC afferent signaling complex (P<0.05). Some GABAergic terminals colocalized with NMDA receptors at the IHC nucleus region (P>0.05). CONCLUSION: There is significant difference in the distribution of NMDA and AMPA receptors in cochlear afferent signaling complex. NMDA receptors are present in the extra-synaptic region of ribbon synapses of IHCs, and they are related to GABA efferent terminals of the afferent signaling complex.

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.

Chromatin remodeling protein CHD4 regulates axon guidance of spiral ganglion neurons in developing cochlea.

The chromodomain helicase binding protein 4 (CHD4) is an ATP-dependent chromatin remodeler. De-novo pathogenic variants of CHD4 cause Sifrim-Hitz-Weiss syndrome (SIHIWES). Patients with SIHIWES show delayed development, intellectual disability, facial dysmorphism, and hearing loss. Many cochlear cell types, including spiral ganglion neurons (SGNs), express CHD4. SGNs are the primary afferent neurons that convey sound information from the cochlea, but the function of CHD4 in SGNs is unknown. We employed the Neurog1(Ngn1) CreERT2 Chd4 conditional knockout animals to delete Chd4 in SGNs. SGNs are classified as type I and type II neurons. SGNs lacking CHD4 showed abnormal fasciculation of type I neurons along with improper pathfinding of type II fibers. CHD4 binding to chromatin from immortalized multipotent otic progenitor-derived neurons was used to identify candidate target genes in SGNs. Gene ontology analysis of CHD4 target genes revealed cellular processes involved in axon guidance, axonal fasciculation, and ephrin receptor signaling pathway. We validated increased Epha4 transcripts in SGNs from Chd4 conditional knockout cochleae. The results suggest that CHD4 attenuates the transcription of axon guidance genes to form the stereotypic pattern of SGN peripheral projections. The results implicate epigenetic changes in circuit wiring by modulating axon guidance molecule expression and provide insights into neurodevelopmental diseases.

Cobalt-induced apoptosis of cochlear organotypic cultures and HEI-OC1 cells is mediated by Dicer.

Cobalt is widely used in the medical industry, mainly including cobalt alloy joint implants and cobalt-chromium porcelain crowns. However, unexplained ototoxicity and neurotoxicity often occur in the clinical use of cobalt agents at present, which limits the development of the cobalt industry. In this study, based on the clinical problem of cobalt ototoxicity, we first conducted an extensive search and collation of related theories, and on this basis, prepared an HEI-OC1 cell model and basilar membrane organotypic cultures after cobalt treatment. We used immunofluorescence staining, western blot, CCK8, and si-RNA to investigate the mechanism of cobalt ototoxicity, to discover its potential therapeutic targets. After comparing the reactive oxygen species, mitochondrial transmembrane potential, apoptosis-related protein expression, and cell viability of different treatment groups, the following conclusions were drawn: cobalt causes oxidative stress in the inner ear, which leads to apoptosis of inner ear cells; inhibition of oxidative stress and apoptosis can alleviate the damage of cobalt on inner ear cells; and the Dicer protein plays a role in the mechanism of inner ear damage and is a potential target for the treatment of cobalt-induced inner ear damage. Taken together, these results suggest that cobalt-induced ototoxicity triggered by oxidative stress activates a cascade of apoptotic events where cCaspase-3 decreases Dicer levels and amplifies this apoptotic pathway. It may be possible to prevent and treat cobalt ototoxicity by targeting this mechanism.

Mechanisms of age-related hearing loss at the auditory nerve central synapses and postsynaptic neurons in the cochlear nucleus.

Sound information is transduced from mechanical vibration to electrical signals in the cochlea, conveyed to and further processed in the brain to form auditory perception. During the process, spiral ganglion neurons (SGNs) are the key cells that connect the peripheral and central auditory systems by receiving information from hair cells in the cochlea and transmitting it to neurons of the cochlear nucleus (CN). Decades of research in the cochlea greatly improved our understanding of SGN function under normal and pathological conditions, especially about the roles of different subtypes of SGNs and their peripheral synapses. However, it remains less clear how SGN central terminals or auditory nerve (AN) synapses connect to CN neurons, and ultimately how peripheral pathology links to structural alterations and functional deficits in the central auditory nervous system. This review discusses recent progress about the morphological and physiological properties of different subtypes of AN synapses and associated postsynaptic CN neurons, their changes during aging, and the potential mechanisms underlying age-related hearing loss.

Sal idroside Modulates cAMP/PKA/CREB Signaling Pathway to Inhibit Ci splatin-induced Damages of Cochlear Hair Cells and Spiral Ganglion Neurons in Mice / 听力学及言语疾病杂志

Objective To investigate the ameliorating effect of salidroside(SAL)on cisplatin(CIS)-induced damages of cochlear hair cells(CHC)and spiral ganglion neurons(SGNs)and its relationship with cyclic adenosine monophosphate(cAMP)/protein kinase A(PKA)/cAMP response element binding protein(CREB)pathway.Methods The cochlear basilar membranes of newborn C 57BL/6 mice were isolated and separated into control(C)group,CIS group,SAL group,SAL+SQ22536(cAMP inhibitor)group and SAL+H-89(PKA inhibitor)group,20 per group.Immunofluorescence staining was applied to observe the damages of CHC and SGNs.The kits were applied to detect the contents of ROS and cAMP in the basement membrane of the cochlea.Western blot was applied to detect the protein levels of PKA,p-CREB,CREB,Bcl-2,BDNF,and NF-M.Results CHC in CIS group were disorderly arranged and enlarged in size,SGNs had fragmented nuclei and lost neurites.SAL alleviated the damages of CHC and SGNs.Compared with the C group,the numbers of CHC and SGNs in the CIS group were less(P＜0.05),the contents of ROS and cAMP,and the levels of PKA,BDNF,NF-M,Bcl-2 proteins and p-CREB/CREB were higher(P＜0.05).Compared with the CIS group,the numbers of CHC and SGNs in the SAL group were higher(P＜0.05),the content of ROS was lower(P＜0.05),the content of cAMP,and the levels of PKA,BD-NF,NF-M,Bcl-2 proteins and p-CREB/CREB were higher(P＜0.05).Both SQ22536 and H-89 reversed the pro-tective effects of SAL on CHC and SGNs.Conclusion SAL may promote the expression of anti-apoptotic proteins and neuroprotective factors by activating the cAMP/PKA/CREB pathway to alleviate the damages of CHC and SGNs caused by CIS.

Protective effects of Ginkgo biloba extract on presbycusis in the rat model via autophagy pathway / 中成药

AIM To investigate the effects of Ginkgo biloba extract on hearing function,cochlear morphology and autophagy-related protein expression in a rat model of presbycusis.METHODS Forty-five rats were randomly divided into the control group,the model group and the low,medium and high dose G.biloba extract groups(10,20 and 30 mg/kg),with 9 rats in each group.The rat model of presbycusis was established by intraperitoneal injection of 500 mg/kg D-galactose(D-gal).Eight weeks after the corresponding administration,the rats had their changes of hearing threshold detected by the auditory brainstem evoked potential(ABR);their morphological changes of cochlear hair cells,stria vascularis(SV)and spiral ganglion cells observed by HE staining;their number of hair cells inside and outside the cochlea detected by immunofluorescence staining;their ultrastructure changes of cochlear hair cells observed by transmission electron microscopy;and their expression of autophagy-related proteins in cochlea tissue detected by Western blot.RESULTS Compared with the control group,the model group displayed increased ABR threshold(P<0.01);more severely damaged inner and outer hair cells,spiral ganglion cells and SV,decreased SV thickness and numbers of spiral ganglion cells,inner and outer hair cells and autophagosomes(P<0.01);decreased protein expressions of Beclin1 and LC3 Ⅱ and ratio of LC3 Ⅱ/LC3 Ⅰ in cochlear tissue(P<0.01),and higher P62 protein expression(P<0.01).Compared with the model group,the medium and high dose G.biloba extract groups shared decreased ABR thresholds(P<0.01);improved morphology of inner and outer hair cells and SV in the cochlea,normalized,morphology of spiral ganglion cells,and increased SV thickness and the numbers of spiral ganglion cells,inner and outer hair cells and autophagosomes(P<0.05,P<0.01);increased protein expressions of Beclin1 and LC3 Ⅱ and the ratio of LC3 Ⅱ/LC3 Ⅰ in the cochlea(P<0.01),and decreased P62 protein expression(P<0.01).CONCLUSION The protective effects G.biloba extract on hearing function and cochlear cells in the rat model of presbycusis may be associated with the up-regulated expression of Beclin1 and LC3 Ⅱ proteins and down-regulated P62 protein expression in cochlear tissues.

CMV-induced Hearing Loss.

Congenital cytomegalovirus (cCMV) infection is the most common fetal viral infection and contributes to about 25% of childhood hearing loss by the age of 4 years. It is the leading nongenetic cause of sensorineural hearing loss (SNHL). Infants born to seroimmune mothers are not completely protected from SNHL, although the severity of their hearing loss may be milder than that seen in those whose mothers had a primary infection. Both direct cytopathic effects and localized inflammatory responses contribute to the pathogenesis of cytomegalovirus (CMV)-induced hearing loss. Hearing loss may be delayed onset, progressive or fluctuating in nature, and therefore, a significant proportion will be missed by universal newborn hearing screening (NHS) and warrants close monitoring of hearing function at least until 5-6 years of age. A multidisciplinary approach is required for the management of hearing loss. These children may need assistive hearing devices or cochlear implantation depending on the severity of their hearing loss. In addition, early intervention services such as speech or occupational therapy could help better communication, language, and social skill outcomes. Preventive measures to decrease intrauterine CMV transmission that have been evaluated include personal protective measures, passive immunoprophylaxis and valacyclovir treatment during pregnancy in mothers with primary CMV infection. Several vaccine candidates are currently in testing and one candidate vaccine in phase 3 trials. Until a CMV vaccine becomes available, behavioral and educational interventions may be the most effective strategy to prevent maternal CMV infection.

Effect of Kidney-nourishing,Blood-activating,Phlegm-resolving and Resuscitation-inducing Decoction on Expression of NMDA Receptor Subtypes in Cochlear Spiral Ganglion Neurons of Tinnitus Rats / 广州中医药大学学报

Objective To observe the effect of Kidney-nourishing, Blood-activating, Phlegm-resolving and Resuscitation-inducing Decoction(KBPRD)on the expression of N-methyl-D-aspartate(NMDA)receptor subtype 1,2A,2B(NR1,NR2A,NR2B)in the cochlear spiral ganglion neurons(SGN)of tinnitus rats and to explore its mechanism, thus to provide experimental evidence for the treatment of tinnitus with KBPRD. Methods Sixty rats were randomly divided into normal group,model group,western medicine group,and low-,middle-,and high-dose Chinese medicine groups, 10 rats in each group. Rats were given intraperitoneal injection of sodium salicylate combined with water deprivation to induce tinnitus model. After successful establishment of the model, the rats in low-,middle-,and high-dose Chinese medicine groups were given gastric administration of KBPRD in the dosage of 5.5, 11, 22 g·kg-1·d-1 respectively, the rats in western medicine group were given gastric administration of 5 mg·kg-1·d-1 of carbamazepine,and rats in the model group and normal group were given gastric administration of 2 mL of normal saline,once every day,treatment time covering 8 weeks. The expression levels of NR1,NR2A,and NR2B in the cochlear SGN was detected by immunoblotting and real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)after 8 weeks of treatment. Results Compared with the normal group,the expression levels of NR1, NR2A and NR2B in the model group were increased, the difference being significant (P < 0.05). Compared with the model group,the expression of NR1,NR2A and NR2B in low-,middle-,and high-dose Chinese medicine groups were significantly decreased(P<0.05).Conclusion KBPRD is effective on relieving tinnitus of rats, and its mechanism is correlated with lowering the increased expression of NR1,NR2A and NR2B in SGN of tinnitus rats.

Correlation of PDCD5 and Apoptosis in Hair Cells and Spiral Ganglion Neurons of Different Age of C57BL/6J Mice / 华中科技大学学报（医学）（英德文版）

This study examined the expression pattern of programmed cell death 5 (PDCD5) in cochlear 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.

The Effect of Ginkgo Biloba on the Survival of Spiral Ganglion Neurons in Rats / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: Ginkgo biloba extract (GBE) enhances cell survival in various organs. GBE protects nerve cells in the central nervous system and is clinically applied in Parkinson's and Alzheimer's disease. GBE can protect ototoxicity caused by cisplantin and gentamycin through rescue of hair cells in Organ of Corti and is accepted as one of the therapeutic agents for sudden deafness and tinnitus. The experimental study on GBE for the inner ear is confined to the hair cells, not to the spiral ganglion neurons (SGNs) which is the stimulated part by the electrode of cochlear implant. The aim of this study is to elucidate the effect of GBE on the survival of SGNs after hair cell loss in rats. MATERIALS AND METHOD: Ten Sprague-Dawley rats aged 50 days (P50) were deafened with kanamycin sulfate. GBE (EGb 761) was injected into the right cochlea and artificial perilymph was injected into the left side. The number and size of SGNs were compared after immunohistochemical statin in both groups. The expression of pJun, which is well-known as a proapoptotic transcription factor in the cochlea, was also compared. RESULTS: The number of SGNs was significantly larger in the GBE group than the control. The expression of pJun activity was significantly decreased in GBE group than the control. The size of SGNs in both groups was similar. CONCLUSION: These results suggest that GBE can protect SGNs death by inhibiting the pJun-C-jun N-terminal kinase pathway. GBE might be a potential drug for the patients with total deafness before or after cochlear implantation surgery for better hearing results.

Electrophisiologic study of routine spiral ganglion neurons / 临床耳鼻咽喉头颈外科杂志

Objective: To explore the nature of voltage dependent ion channels and basic electrophysiological characteristics of cochlear spiral ganglion neurons of apical and basal turn by patch clamp techniques of whole cell configure on murine spiral ganglion neurons. Method: Different voltage dependent ionic currents were recorded with patch clamp techniques of whole cell configure on the condition of different internal electrode solution,blockers and stimulus protocol. Result: Inward sodium channel current(I_(Na)), hyperpolarization-activated inward cationic current(I_h) ,outward delay rectification potassium current (I_k) and outward transient potassium current (I_A)were recorded , significant difference of electrophysiological characteristics of I_A and I_k was found between apical and basal turns(P＜0.05). Conclusion: Various ionic currents are recorded,which shows that spiral ganglion neurons have the base of ionic channels to complete formation,conduction and modulation of action potential for auditory information transduction, the difference of electrophysiological characteristics between apical and basal turns contributes to the course of hearing formation.

Preliminary Study on Firing Patterns of Murine spiral Ganglion Neurons by Patch-clamp Technique / 听力学及言语疾病杂志

Objective To investigate the fire patterns of the primary cultured postnatal mouse spiral ganglion neurons.Methods Current-clamp recordings with the use of the whole cell configuration of the patch-clamp technique were made from the primary cultured spiral ganglion neurons. Results The action potential was recorded from the spiral ganglion neurons. The neurons had non-uniform firing characteristics. Conclusion There were two firing patterns observed in the spiral ganglion neurons, rapid adaptation, and slow adaptation and the largest neurons showing rapidly adapting.