Spiral ganglion neurons (SGNs) transmit auditory information from cochlear hair cells to the brain. SGNs are thus not only important for normal hearing, but also for effective functioning of cochlear implants, which stimulate SGNs when hair cells are missing. SGNs slowly degenerate following aminoglycoside-induced hair cell loss, a process thought to involve an immune response. However, the specific immune response pathways involved remain unknown. We used RNAseq to gain a deeper understanding immune-related and other transcriptomic changes that occur in the rat spiral ganglion after kanamycin-induced deafening. Among the immune and inflammatory genes that were selectively upregulated in deafened spiral ganglia, the complement cascade genes were prominent. We then assessed SGN survival, as well as immune cell numbers and activation, in the spiral ganglia of rats with a CRISPR-Cas9-mediated knockout of complement component 3 (C3). Similar to previous findings in our lab and other deafened rodent models, we observed an increase in macrophage number and increased expression of CD68, a marker of phagocytic activity and cell activation, in macrophages in the deafened ganglia. Moreover, we found an increase in MHCII expression on spiral ganglion macrophages and an increase in lymphocyte number in the deafened ganglia, suggestive of an adaptive immune response. However, C3 knockout did not affect SGN survival or increase in macrophage number/activation, implying that complement activation does not play a role in SGN death after deafening. Together, these data suggest that both innate and adaptive immune responses are activated in the deafened spiral ganglion, with the adaptive response directly contributing to cochlear neurodegeneration.

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Ototoxicity is a major side effect of aminoglycosides, which can cause irreversible hearing loss. Previous studies on aminoglycoside-induced ototoxicity have primarily focused on the loss of sensory hair cells. Recent investigations have revealed that aminoglycosides can also lead to the loss of ribbon synapses in inner hair cells (IHCs). However, the functional implications of ribbon synapse loss and the underlying mechanisms remain unclear. In this study, we intraperitoneally injected C57BL/6 J mice with 300 mg/kg gentamicin once daily for 3, 10, and 20 days. Then, we performed immunofluorescence staining, patch-clamp recording, proteomics analysis and western blotting to characterize the changes in ribbon synapses in IHCs and the associated mechanisms. After gentamicin treatment, the auditory brainstem response (ABR) threshold was elevated, and the ABR wave I amplitude was decreased. We also observed loss of ribbon synapses in IHCs. Interestingly, ribbon synapse loss occurred on both the modiolar and pillar sides of IHCs. Whole-cell patch-clamp recordings in IHCs revealed a reduction in the calcium current amplitude, along with a shifted half-activation voltage and altered calcium voltage dependency. Moreover, exocytosis of IHCs was reduced, consistent with the reduction in the ABR wave I amplitude. Through proteomic analysis, western blotting, and immunofluorescence staining, we found that gentamicin treatment resulted in downregulation of myosin VI, a protein crucial for synaptic vesicle recycling and replenishment in IHCs. Furthermore, we evaluated the kinetics of endocytosis and found a significant reduction in IHC exocytosis, possibly reflecting the impact of myosin VI downregulation on synaptic vesicle recycling. In summary, our findings demonstrate that gentamicin treatment leads to synaptic dysfunction in IHCs, highlighting the important role of myosin VI downregulation in gentamicin-induced synaptic damage.

The Toll-like receptor (TLR) signaling pathway is the key regulator of the innate immune system in response to systemic infection. Several studies have reported that the systemic TLR4 agonist lipopolysaccharide exacerbates aminoglycoside ototoxicity, but the influence of virus-associated TLR7 and TLR9 signaling cascades on the cochlea is unclear. The present study aimed to investigate the auditory effects of systemic TLR7 and TLR9 agonists during chronic kanamycin treatment. CBA/CaJ mice received the TLR7 agonist gardiquimod or TLR9 agonist CpG oligodeoxynucleotides (ODN) one day before kanamycin injection and on the 5th and 10th days during a 14-day course of kanamycin treatment. We observed that systemic gardiquimod or CpG ODN alone did not affect the baseline auditory brainstem response (ABR) threshold. Three weeks after kanamycin treatment, gardiquimod did not significantly change ABR threshold shifts, whereas CpG ODN significantly increased kanamycin-induced ABR threshold shifts. Furthermore, outer hair cell (OHC) evaluation revealed that CpG ODN reduced distortion product otoacoustic emission amplitudes and increased kanamycin-induced OHC loss. CpG ODN significantly elevated cochlear Irf-7, Tnf-α, Il-1, and Il-6 transcript levels. In addition, an increased number of Iba-1+ cells, which represented activated macrophages, was observed in the cochlea treated with CpG ODN. Our results indicated that systemic CpG ODN exacerbated kanamycin-induced ototoxicity and increased cochlear inflammation. This study implies that patients with underlying virus infection may experience more severe aminoglycoside-induced hearing loss if it occurs.

Purinergic signaling regulates important physiological processes and the homeostatic response to stress in the cochlea via extracellular nucleosides (adenosine) and nucleotides (ATP, UTP). Using a previously established organotypic culture model, the current study investigated the effect of purinergic P1 (adenosine) and P2 (ATP) receptor activation on the survival of the sensory hair cell population in the cochlea exposed to the ototoxic aminoglycoside neomycin. Organ of Corti explants were obtained from C57BL/6 mice at postnatal day 3 (P3) and maintained in normal culture medium (with or without purine receptor agonists or analogs) for 19.5 h prior to neomycin exposure (1 mM, 3 h) followed by a further incubation for 19.5 h in culture medium. The cochlear explants were then fixed in 4% paraformaldehyde (PFA) and sensory hair cells labeled with Alexa 488-phalloidin. Neomycin induced a substantial loss of the sensory hair cells, mostly in the middle segment of the cochlea. This neomycin-induced ototoxicity was unaffected by the addition of P2 receptor agonists (ATP and UTP) in the culture medium, whilst the addition of their slowly-hydrolyzable analogs (ATPγS, UTPγS) aggravated neomycin-induced sensory hair cell loss. In contrast, the activation of P1 receptors by adenosine or adenosine amine congener (ADAC) conferred partial protection from neomycin ototoxicity. This study demonstrates a pro-survival effect of P1 receptor stimulation, whilst prolonged activation of P2 receptors has an opposite effect. Based on these findings, we postulate that P1 and P2 receptors orchestrate differential responses to cochlear injury and that the balance of these receptors is important for maintaining cochlear homeostasis following ototoxic injury.

Aminoglycoside ototoxicity results in permanent loss of the sensory hair cells in the mammalian cochlea. It usually begins at the basal turn causing high-frequency hearing loss. Here we describe previously unreported resistance of hair cells to neomycin ototoxicity in the extreme basal (hook) region of the developing cochlea of the C57BL/6 mouse. Organ of Corti explants from mice at postnatal day 3 were incubated (37 °C, 5% CO2) in normal culture medium for 19.5 h prior to and after exposure to neomycin (1 mM, 3 h). To study neomycin uptake in the hair cells, cochlear explants were incubated with Neomycin Texas-red (NTR) conjugate. As expected, exposure to neomycin significantly reduced the survival of inner (IHC) and outer hair cells (OHC). IHC survival rate was high in the apical segment and low in the basal segment. OHC were well preserved in the apical and hook regions, with substantial OHC loss in the basal segment. The NTR uptake study demonstrated that the high survival rate in the extreme basal turn OHC was associated with low NTR uptake. Treatment with a calcium chelator (BAPTA), which disrupts the opening of mechanoelectrical (MET) transduction channels, abolished or reduced NTR uptake in the hair cells throughout the cochlea. This confirmed the essential role of MET channels in neomycin uptake and implied that the transduction channels could be impaired in the hook region of the developing mouse cochlea, possibly as a result of the cadherin 23 mutation responsible for the progressive deafness in C57BL/6 mice.

Effective management of patients diagnosed with ototoxicity is needed to reduce hearing and balance damage which affects communication and life quality. Despite widespread recommendations to monitor and manage ototoxicity in an early and effective manner, there is limited evidence to support the actual implementation of these recommendations for affected patient groups in healthcare services across the UK with limited publications available. In this study, an online questionnaire analysed the current practice of ototoxicity management and patient pathways across the UK once the diagnosis of ototoxicity was confirmed, targeting Audiologists, ENTs/AVPs and GPs.
Qualitative Survey Study.
A randomised sample of hearing services in the UK, including audiology departments; GP practices and local health settings were targeted with a total of 134 completed surveys.
About 72% reported the absence of ototoxicity management protocols within their centre. Results depicted great inconsistency and variation across the UK in ototoxicity management services provided, treatment modification, monitoring and referral pathways.
Developing and advocating national guidelines are intended not only to inform clinical decision making but to provide minimum standards of care in ototoxicity management and offer greater awareness and education to improve patients\' quality of life.

Many previous studies have shown significant neurotrophic effects of intracochlear delivery of BDNF in preventing degeneration of cochlear spiral ganglion (SG) neurons after deafness in rodents and our laboratory has shown similar results in developing cats deafened prior to hearing onset. This study examined the morphology of the cochlear nucleus (CN) in a group of neonatally deafened cats from a previous study in which infusion of BDNF elicited a significant improvement in survival of the SG neurons. Five cats were deafened by systemic injections of neomycin sulfate (60 mg/kg, SQ, SID) starting one day after birth, and continuing for 16-18 days until auditory brainstem response (ABR) testing demonstrated profound bilateral hearing loss. The animals were implanted unilaterally at about 1 month of age using custom-designed electrodes with a drug-delivery cannula connected to an osmotic pump. BDNF (94 μg/ml; 0.25 μl/hr) was delivered for 10 weeks. The animals were euthanized and studied at 14-23 weeks of age. Consistent with the neurotrophic effects of BDNF on SG survival, the total CN volume in these animals was significantly larger on the BDNF-treated side than on the contralateral side. However, total CN volume, both ipsi- and contralateral to the implants in these deafened juvenile animals, was markedly smaller than the CN in normal adult animals, reflecting the severe effects of deafness on the central auditory system during development. Data from the individual major CN subdivisions (DCN, Dorsal Cochlear Nucleus; PVCN, Posteroventral Cochlear Nucleus; AVCN, Anteroventral Cochlear Nucleus) also were analyzed. A significant difference was observed between the BDNF-treated and control sides only in the AVCN. Measurements of the cross-sectional areas of spherical cells showed that cells were significantly larger in the AVCN ipsilateral to the implant than on the contralateral side. Further, the numerical density of spherical cells was significantly lower in the AVCN ipsilateral to the implant than on the contralateral side, consistent with the larger AVCN volume observed with BDNF treatment. Together, findings indicate significant neurotrophic effects of intracochlear BDNF infusion on the developing CN.