Introduction

Spiral ganglion neurons (SGNs) in the cochlea are the primary afferent auditory neurons of information.[1–3] They play an important role in the integration and delivery of sound arriving at cochlear hair cells (HCs). Human SGNs are divided into two types, type I and type II. Type I SGNs account for 90%–95% of the total number of SGNs, while type II account for 5%–10%.[4] Type I SGNs have large cell bodies and bipolar neurites that associate mainly with the inner hair cells (IHCs) of the organ of Corti via synapses.[1,5] Type II SGNs have smaller bodies and shorter axons than type I neurons, and there is more variability in their shape as either bipolar or psuedounipolar. They emit central projections to the granule cell layer in the cochlear nucleus and peripheral projections to the outer hair cells (OHCs) by way of synapses.[4,6] A minimum number of functional SGNs is essential to maintain normal auditory function, and injury to SGNs directly affects auditory function. Aging, ototoxic drugs, and intense sound/noise, can lead to apoptosis of SGNs and affect auditory function, resulting in hearing loss.[7] It has been reported that neurotrophic factors (NTFs), estrogen, electrical stimulation, and stem cell transplantation (SCT) may promote the survival of SGNs and regrowth of neurites. A better understanding of the mechanisms of injury and the protection, and regeneration of SGNs will aid in the design of potential therapeutic strategies for patients suffering from hearing loss related to SGN injury. In this paper, we cover the present knowledge concerning these aspects of SGNs.

Injury Mechanisms to SGNs Ototoxic drugs Aminoglycoside antibiotics (AmAn)

AmAn primarily damage the sensory HCs of the cochlea and vestibule, causing irreversible sensorineural hearing loss (SNHL) and balance dysfunction. SGNs, which directly accept cochlear HC afferent signals, experience degeneration because of the lack of stimulation from the neuron and lack of NTFs secondary to permanent damage to the HC. It is through this mechanism that secondary and delayed death of SGNs occurs. Following entry via ion channels into the cytoplasm, aminoglycosides bind to hundreds of proteins. Aminoglycosides also bind to the phosphatidylinositol family of lipids, particularly phosphatidylinositol 4,5-bisphosphatem (PIP2) which, in mammalian HCs, blocks voltage-gated channels, outwardly rectifying potassium channels on the basolateral membranes of OHCs. This blockade prevents the rapid repolarization of HCs crucial for their survival.[8] However, HCs are not the only target of AmAn. Supporting cells in the organ of Corti may be implicated as well. For example, damage to HCs is frequently accompanied by the injury to the inner phalangeal cells. Ding et al[9] suggest that AmAn cause damage to HCs and cochlear supporting cells simultaneously, and this eventually leads to the delayed death of SGNs. In addition, the neurogliocytes around the SGNs dedifferentiate injury from AmAn, causing loss of the NTFs, which accelerate delayed neuronal death.

Kanamycin and gentamicin, among other AmAn, do not pass through the blood-labyrinth barrier (BLB) easily. Instead, it is thought that they damage the auditory system through chronic accumulation in the inner ear fluids and then specifically attack inner ear HCs and the surrounding supporting cells gradually, rather than damaging the sensory neurons around the inner ear directly.

Streptomycin, which represents another category of AmAn, passes easily through the BLB and damages the auditory nerve specifically. It can instantly and directly damage the afferent and efferent nervous system in the inner ear, while causing inconspicuous damage of the inner ear sensory HCs in the early stage of the disease. Gao et al[9] found that the streptomycin sulfate's toxic effect to the auditory afferent system mainly manifests in the inhibition of the postsynaptic potential, the prolonged latency of acoustic nerve action potential, and prolonged latency and interwave period of auditory brainstem response.

Sodium salicylate (SS)

While the exact mechanism behind salicylate-induced hearing impairment is unclear, there is evidence to suggest that salicylate mainly eliminates OHC electromotility and influences cochlear blood flow.[10] High doses of salicylate may result in hearing loss and tinnitus temporarily by impairing neuronal function at multiple sites along the auditory pathway beginning with the OHCs and SGNs in the cochlea and progressing centrally to the auditory cortex (AC). Feng et al[11] demonstrated that long-term treatment with high doses of salicylate can exert neurotoxic degeneration effects on adult SGNs in vitro. Salicylate damages SGNs and their peripheral fibers in a dose-dependent manner, rather than damaging sensory HCs directly.

In a previously published paper, Feng et al[11] determined that salicylate could induce apoptosis in the SGN and auditory HCs in association with caspase-3 activation. Another more recent study suggests that a caspase-mediated cell death pathway, focusing on SGNs apoptosis, is involved in salicylate-induced ototoxicity. Long-term administration of high doses of salicylate induces a cascade reaction to the primary auditory neuron, where it acts to induce apoptosis in a caspase-dependent manner. High doses of salicylate in a physiologically relevant range can induce caspase-mediated cell death in immature SGNs; changes in the expression of apoptotic genes particularly among members of the tumor necrosis factor (TNF) family appear to play an important role in the degeneration. The results of the study of Huang et al[12] showed that SS-induced ototoxicity was significantly correlated with the high expression levels of receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like protein (MLKL), reflecting the necrotic cell death pathway.

A recent study has found that neuroinflammation may be a new mechanism of injury caused by SS.[13] Zhai et al[13] suggested that, in the process of hearing injury of animal models, SS may regulate downstream caspase-1 and interleukin-1β (IL-1β) transcription by activating nod-like receptor protein 3 (NLRP3) inflammasome in auditory SGNs, thus damaging the auditory system and causing hearing loss.

Cisplatin

Cisplatin is widely used in the treatment of various malignant tumors as a chemotherapeutic agent. However, it can cause serious ototoxic side effects including SNHL, ranging from mild to severe. Sun et al[14] suggested that the ototoxic damage of cisplatin proceeds in order from high frequencies to low frequencies. In the early stages, cisplatin may cause damage through affecting the stria vascularis, but such damage is reversible. When cisplatin administration continues, it can cause irreversible morphological damage to cochlea, resulting in high-frequency SNHL. Cisplatin affects many intracellular pathways and binds to hundreds of proteins that can potentially dysregulate cells and lead to cell death. For cochlear cells, uptake of cisplatin triggers the transcription factor, signal transducer, and activator of transcription 1 (STAT1) to activate the transient receptor potential vanilloid subtype 1 (TRPV1) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 3 (NOX3) signaling pathways that generate toxic levels of reactive oxygdtimately to induce cell death.[8]

Alam et al[15] confirmed that terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labelling (TUNEL)-positive cells appear in the OHCs and spiral ganglion cells after cisplatin injection. By establishing a mouse ototoxicity model of cisplatin, Sun et al[14] found that with the increasing dose of cisplatin, the number of TUNEL-positive cells in mouse cochlear spiral ganglion increased, and the enhanced expression of caspase-3 indicated that cisplatin could lead to the apoptosis of cochlear spiral ganglion cells, which was further confirmed by the involvement of caspase-3, meaning that apoptosis is one of the mechanisms of cisplatin injury. Zhang et al[16] observed the apoptosis of a large number of spiral ganglion cells in the cochlea of guinea pigs after cisplatin application, while the expression level of caspase-3 was significantly increased, suggesting that caspase-3 was activated in the process of cisplatin-induced apoptosis of spiral ganglion cells.

The apoptotic protein Bcl-2 19-kDa interacting protein 3 (BNIP3) also plays an important role in cisplatin-induced spiral ganglion cell death. BNIP3 follows an independent signal transduction pathway and enters the nucleus to bind with DNA and upregulate apoptotic gene expression to promote cell apoptosis. It has been observed that the positive expression of caspase-3 is found in the spiral ganglion of cochlea of mice in each group after the application with different doses of cisplatin, mainly located in the cytoplasm, with the expression of caspase-3 significantly enhanced with increasing doses of cisplatin. These results indicate that caspase, as the central link and executor of apoptosis, is also involved in the ototoxic injury process of cisplatin in mice, further confirming that cochlear cell apoptosis may be one of the ototoxic mechanisms of cisplatin.

Glutamic acid

Excessive intracellular calcium accumulation is another common mechanism behind cell death, leading eventually to cell degeneration, necrosis, or apoptosis. Yan et al[17] found that under the effect of ischemia, hypoxia, or noise, glutamic acid is excessively released or insufficiently reuptaken, and the N-methyl-D-aspartate (NMDA) receptor is excessively activated or the activity of NMDA receptor is overenhanced, leading to an excess of calcium ions in the spiral ganglion cells. This leads to damage to SGNs. Yan et al[17] showed that 24 h after exposure to glutamic acid, most of the spiral ganglion cells were damaged, showing morphological changes of apoptosis or death. At the same time, it was observed that the concentration of free calcium ions in spiral ganglion cells increased rapidly after the addition of glutamic acid, indicating that glutamate-receptor-dependent calcium channels exist on spiral ganglion cells, and certain concentration of glutamic acid can cause the overloading of calcium in spiral ganglion cells.

Noise

Noise-induced hearing loss (NIHL) is a common occupational injury. Noise exposures causing large but reversible/temporary threshold shifts (TTSs) can nevertheless cause rapid (occurring in minutes to hours) loss of up to 50% of the synapses between cochlear neurons and the IHCs they contact.[18] Noise can damage SGNs, and previous studies have suggested that the acoustic damage mechanism of SGNs is related to the neurotoxic effect caused by the excessive release of neurotransmitter glutamic acid by HCs. Chen et al[19] suggest that one of the acoustic injury mechanisms of SGNs may be related to the overloading of Ca2+. Ca2+ combines calmodulin (CaM) to activate various factors which lead to neuron injury. It can be inferred that CaM plays an important role in the acoustic injury mechanism of SGNs. In Chen's study, it was found that CaM expression of SGNs in guinea pig cochlea with NIHL was significantly increased, and this increase tended to be significantly increased with the aggravation of NIHL, related to the degree of acoustic damage of SGNs. They speculated that the increasing expression of CaM in the acoustic injury of SGNs was related to the stress response degree of SGNs, and it was considered that the CaM level of SGNs could be used as an index to evaluate the degree of the acoustic injury of SGNs.

Huang et al[20] found that apoptosis of SGNs can be induced by intense noise and the caspase-3 cascade may be involved in the course of SGNs apoptosis. SGNs may be affected by noise, resulting in cumulative and non-recoverable damage. Part of the damage occurred after the death of the connected HCs, and part of the damage was unrelated to the HCs but along with the extinction and degeneration of SGNs, ultimately leading to hearing loss. Exposure to loud noise can induce SGNs by inducing apoptosis protease and thus promoting cell apoptosis. Experiments have shown that SGNs apoptosis is closely related to the activation of caspase-3.[20]

Aging and presbycusis

Age-related hearing loss–presbycusis–is the number one communication disorder and most prevalent neurodegenerative condition of the aging population. Presbycusis arises from irreversible damage in the inner ear, where sound is transduced into electrical signals.[21] The expression of the neurotrophins brain-derived neurotrophic factor (Bdnf) and neurotrophin factor-3 (NTF3) is needed for maintaining SGNs, and in their absence, no SGNs survive.[7] The loss of HCs and degeneration of SGNs results in high-frequency hearing impairment, progressing to impairment across all frequencies, together known as presbycusis. Tang et al[22] revealed that SGNs decrease with age in the apical, middle, and basal regions, with the basal area most affected.

Tang et al[22] discovered that gammaaminobutyric acid type A receptor α1 (GABAARα1) subunit expression downregulates with age, consistent with the concept that the normal protective action of gamma-aminobutyric acid type A receptors (GABAARs) in the cochlea for HCs and SGNs survival is less effective with aging, thus contributing to the deleterious effects of age-related hearing loss in the inner ear. SGNs express α2, α4-7, and β2-3 nicotinic acetylcholine receptor (nAChR) subunits. Mice lacking the nAChR subunit (β2-/-) have dramatic hearing loss and significant reduction in the number of SGNs. Similarly, further research showed that nAChR β2 expression decreased in the presbycusis mouse model, likely contributing to functional hearing changes during aging. Moreover, genetic mutations can also predispose one to more rapid age-related cochlear dysfunction; for example, the glutamate receptor metabotropic 7 (GRM7) expressed in HC/SGN synapse has been identified as one of the first presbycusis genes in humans. Furthermore, mitochondrial damage caused by reactive oxygen species (ROS) can induce age-related SGNs deaths via necrosis or apoptosis.

Frisina et al[23] found that presbycusis in the CBA/CaJ mouse involves both HC and SGN loss. Their data indicate that the extrinsic and intrinsic apoptotic pathways are upregulated with age in SGNs. Caspase-3 expression increased in middle-age SGNs as compared with young adults. Also, the apoptotic intrinsic inducer Bax was found to be upregulated, and the survivor factor Bcl-2 was downregulated with aging in SGNs. They suspect that aging initiates apoptosis processing in an inner loop network, which is to inhibit several key molecules, rather than the whole apoptotic pathways.

Protection and Regeneration of SGNs

Prevention or reversal of SGN degeneration is critically important in helping patients with hearing loss. Unfortunately, there are no drug therapies that can reliably protect or restore hearing. Drug delivery to the inner ear via the vasculature is an attractive non-invasive strategy, yet the BLB at the luminal surface of inner ear capillaries restricts the entry of most blood-borne compounds into inner ear tissues. Therefore, the physical inaccessibility of the inner ear and the BLB creates challenges for effective delivery of therapeutics.[24] Elliot et al[25] showed that a long-term loss of Bdnf leads to a significant reduction in the number of vestibular ganglion neurons and a reduction in the number of vestibular HCs. There was no significant decrease in the lateral vestibular nucleus (LVN) or the cerebellum at 6 months, suggesting that the connectivity between central target cells and other neurons suffices to prevent their loss despite vestibular HC and ganglion neuron loss.

Neurons of the inner ear require NTFs for survival. NTFs signal through tyrosine receptor kinase (Trk) protein-tyrosine kinase receptors. In the inner ear, Bdnf and NTF3, which signal through the Ntrk2 (TrkB) and Ntrk3 (TrkC) receptors, respectively, are expressed, and in the absence of either NTFs or receptors, no neurons survive. Elliott et al[25] showed that a long-term loss of Bdnf leads to a significant reduction in the number of vestibular ganglion neurons and a reduction in the number of vestibular HCs, and both NTFs and their receptors are essential and sufficient for vestibular neuron viability.

Rapamycin, a specific inhibitor of mammalian target of rapamycin (mTOR) complex 1, was co-applied with gentamicin to verify the role of mTOR signaling in the study of Guo et al[10]. They observed that the number and length of neurites were significantly increased by rapamycin treatment.

In the research of Huang et al[12], RIPK1/RIPK3/MLKL-mediated programed necrosis was found in SS-induced SGN injury in rats, and necrostatin-1 (Nec-1) inhibited programed necrosis and protected SGNs. Nec-1 is a specific inhibitor of programed necrosis, which may restrain the activity of RIPK1 and block the progression of the programed necrosis. Zhai et al[13] demonstrated that MCC950 (also known as cytokine release inhibitory drug 3 [CRID3]), a novel and selective inhibitor of NLRP3-inflammasome can inhibit the activation of NLRP3 and the downstream inflammatory factors and has a significant effect on salicilate-induced SGN injury. Therefore, they speculate that salicylate-induced cochlea SGN inflammatory cascade can be inhibited by MCC950, striking a balance between anti-inflammatory and pro-inflammatory reactions. SS can induce the up-regulating of BDNF exon IV, VI, and caspase-3 in SGNs. This upregulation is reversed when NMDA receptor blocker is used, suggesting that inhibiting the NMDA receptor non-specifically can antagonize the in vitro spiral ganglion excitatory damage in cells induced by SS.

Applying caspase-3 inhibitors in cochlear perfusion can provide downstream protection against cisplatin ototoxicity, significantly reducing cisplatin-induced HC apoptosis and hearing loss. Frisina et al[23] reported that the rescue of SGNs provides anatomical confirmation that aldosterone has a protective effect on SGN survival with age and provides a basis for the maintenance of high-frequency hearing ability.

Soluble form of receptor for advanced glycation-end products (sRAGEs) can reduce advanced glycation end-product (AGE)-induced apoptosis of SGNs and reduce the expression of RAGE receptors on the membrane, which may be related to the competitive binding of AGEs by sRAGE without corresponding biological effects. Wu et al[21] confirmed that sRAGE (0–150 mg/L) can effectively reduce AGE-induced apoptosis of SGNs. Their results suggest that sRAGE can reduce AGE-RAGE signaling pathway-induced apoptosis. The results of their study showed that sRAGE had no effect on normal SGNs but reduce apoptosis of SGNs and reduce the expression of RAGE on the cell membrane.

Moreover, the simultaneous application of the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD) and the necroptosis inhibitor Nec-1 had a significant effect on SGN protection compared with Z-VAD treatment alone, and this inhibition of necroptosis can be an indispensable method for protecting SGNs from death.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta (TGF-β) family, which were originally identified as a component of bony metabolism but are now implicated in many biological activities as a multifunctional protein. Recent studies have shown that BMP4 can promote the survival of mature SGNs in vitro. Whitlon et al[26] found that exogenous BMP4 could promote the survival of SGNs isolated from postnatal mouse cochlea. At the same time, Waqas et al[27] found that high concentration of exogenous BMP4 could promote neurite growth and increase synaptic density of SGNs in vitro, and BMP4 treatment in vitro may promote the survival of cultured cochlear SGNs and preserve their structure and function.

In the aging process, estrogen deficiency can lead to increased apoptosis of spiral ganglion cells and decreased hearing in C57BL/6J mice, and estrogen therapy can ameliorate this progress, suggesting that estrogen can play a protective role on hearing by inhibiting the apoptosis of spiral ganglion cells in the aging process. Chen et al[28] found that estrogen can downregulate the caspase-3 and Bax messenger ribonucleic acid (mRNA) level of SGN apoptosis protein in aged mice and upregulate the mRNA levels of Bcl-2 anti-apoptosis protein.

With regards to electrical stimulation of SGNs, improved SGN survival has been noted. Several experimental studies have shown that this electrical stimulation, especially when combined with growth factors, has a protective effect on SGNs.[29]

Embryonic stem cells are pluripotent and can differentiate into a variety of cell types. Stem cells are typically classified according to their ability to differentiate toward different cell types, and these cells are proposed to be an important source for spiral ganglion nerve cell regeneration following HC loss. If a transplantation approach is to be successful in treating inner ear injuries, it is of course essential that the transplanted cells not only survive but also migrate to functionally relevant regions and differentiate into an appropriate neuronal cell. Human amniotic fluid mesenchymal stem cells (hAFMSCs) having great pluripotent potential may be used in the future for the regeneration of the spiral ganglion, one of the most vital parts of the inner ear.[30]

Conclusions

SGNs serve as the primary relay element for acoustic signals between HCs and the auditory nucleus. Damage to SGNs appears to be a precursor to hearing loss. Various types of damage can injure SGNs, including ototoxic drugs, glutamic acid, noise exposure, and aging, all of which can lead to severe SNHL. In contrast to peripheral neurons and some sensory neurons, SGNs do not regenerate to any clinically significant extent after damage. Therefore, identification of patterns of SGN injury and subsequent blocking of appropriate apoptosis and necrosis pathways represent an essential step in protecting auditory function. Present research suggests that NTFs, compounds and medications such as estrogen and aldosterone, electrical stimulation, and stem cell transplantation may all promote the survival of SGNs and the growth of neurites, and these possibilities offer a glimpse into therapeutic options for the protection and even regeneration of SGNs.

Funding

This study was supported by a grant from the Beijing Municipal Commission of Science and Technology (Applied technology research and development project) (No. Z191100007619043).

Conflicts of interest

None.

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