Molecular Characteristics of Cisplatin-Induced Ototoxicity and Therapeutic Interventions.

Cisplatin is a commonly used chemotherapeutic agent with proven efficacy in treating various malignancies, including testicular, ovarian, cervical, breast, bladder, head and neck, and lung cancer. Cisplatin is also used to treat tumors in children, such as neuroblastoma, osteosarcoma, and hepatoblastoma. However, its clinical use is limited by severe side effects, including ototoxicity, nephrotoxicity, neurotoxicity, hepatotoxicity, gastrointestinal toxicity, and retinal toxicity. Cisplatin-induced ototoxicity manifests as irreversible, bilateral, high-frequency sensorineural hearing loss in 40-60% of adults and in up to 60% of children. Hearing loss can lead to social isolation, depression, and cognitive decline in adults, and speech and language developmental delays in children. Cisplatin causes hair cell death by forming DNA adducts, mitochondrial dysfunction, oxidative stress, and inflammation, culminating in programmed cell death by apoptosis, necroptosis, pyroptosis, or ferroptosis. Contemporary medical interventions for cisplatin ototoxicity are limited to prosthetic devices, such as hearing aids, but these have significant limitations because the cochlea remains damaged. Recently, the U.S. Food and Drug Administration (FDA) approved the first therapy, sodium thiosulfate, to prevent cisplatin-induced hearing loss in pediatric patients with localized, non-metastatic solid tumors. Other pharmacological treatments for cisplatin ototoxicity are in various stages of preclinical and clinical development. This narrative review aims to highlight the molecular mechanisms involved in cisplatin-induced ototoxicity, focusing on cochlear inflammation, and shed light on potential antioxidant and anti-inflammatory therapeutic interventions to prevent or mitigate the ototoxic effects of cisplatin. We conducted a comprehensive literature search (Google Scholar, PubMed) focusing on publications in the last five years.

The developmental journey of therapies targeting purine receptors: from basic science to clinical trials.

Since the discovery of ATP as an extracellular signalling molecule in 1972, purinergic signalling, mediated by extracellular purines and pyrimidines has been identified in virtually all mammalian tissues and is implicated in regulating fundamental cellular processes. In recent years, there has been an increasing focus on the pathophysiology and potential therapeutic interventions based on purinergic signalling. A vast range of compounds targeting purine receptors are in clinical development, and many more are in preclinical studies, which highlights the fast growth in this research field. As a tribute to Professor Geoffrey Burnstock's legacy in purinergic signalling, we present here a brief review of compounds targeting purine receptors that are in different stages of clinical trials. The review highlights the 50-year journey from basic research on purinergic receptors to clinical applications of therapies targeting purine receptors.

Purinergic Signalling in the Cochlea.

The mammalian cochlea is the sensory organ of hearing with a delicate, highly organised structure that supports unique operating mechanisms. ATP release from the secretory tissues of the cochlear lateral wall (stria vascularis) triggers numerous physiological responses by activating P2 receptors in sensory, supporting and neural tissues. Two families of P2 receptors, ATP-gated ion channels (P2X receptors) and G protein-coupled P2Y receptors, activate intracellular signalling pathways that regulate cochlear development, homeostasis, sensory transduction, auditory neurotransmission and response to stress. Of particular interest is a purinergic hearing adaptation, which reflects the critical role of the P2X2 receptor in adaptive cochlear response to elevated sound levels. Other P2 receptors are involved in the maturation of neural processes and frequency selectivity refinement in the developing cochlea. Extracellular ATP signalling is regulated by a family of surface-located enzymes collectively known as "ectonucleotidases" that hydrolyse ATP to adenosine. Adenosine is a constitutive cell metabolite with an established role in tissue protection and regeneration. The differential activation of A1 and A2A adenosine receptors defines the cochlear response to injury caused by oxidative stress, inflammation, and activation of apoptotic pathways. A1 receptor agonism, A2A receptor antagonism, and increasing adenosine levels in cochlear fluids all represent promising therapeutic tools for cochlear rescue from injury and prevention of hearing loss.

A High-Fat Diet Induces Low-Grade Cochlear Inflammation in CD-1 Mice.

There is growing evidence for a relationship between gut dysbiosis and hearing loss. Inflammatory bowel disease, diet-induced obesity (DIO), and type 2 diabetes have all been linked to hearing loss. Here, we investigated the effect of a chronic high-fat diet (HFD) on the development of inner ear inflammation using a rodent model. Three-week-old CD-1 (Swiss) mice were fed an HFD or a control diet for ten weeks. After ten weeks, mouse cochleae were harvested, and markers of cochlear inflammation were assessed at the protein level using immunohistochemistry and at the gene expression level using quantitative real-time RT-PCR. We identified increased immunoexpression of pro-inflammatory biomarkers in animals on an HFD, including intracellular adhesion molecule 1 (ICAM1), interleukin 6 receptor α (IL6Rα), and toll-like-receptor 2 (TLR2). In addition, increased numbers of ionized calcium-binding adapter molecule 1 (Iba1) positive macrophages were found in the cochlear lateral wall in mice on an HFD. In contrast, gene expression levels of inflammatory markers were not affected by an HFD. The recruitment of macrophages to the cochlea and increased immunoexpression of inflammatory markers in mice fed an HFD provide direct evidence for the association between HFD and cochlear inflammation.

Molecular Mechanisms of Sensorineural Hearing Loss and Development of Inner Ear Therapeutics.

The sense of hearing enables us to enjoy sounds and music and engage with other people [...].

The Link between Gut Dysbiosis Caused by a High-Fat Diet and Hearing Loss.

This review aims to provide a conceptual and theoretical overview of the association between gut dysbiosis and hearing loss. Hearing loss is a global health issue; the World Health Organisation (WHO) estimates that 2.5 billion people will be living with some degree of hearing loss by 2050. The aetiology of sensorineural hearing loss (SNHL) is complex and multifactorial, arising from congenital and acquired causes. Recent evidence suggests that impaired gut health may also be a risk factor for SNHL. Inflammatory bowel disease (IBD), type 2 diabetes, diet-induced obesity (DIO), and high-fat diet (HFD) all show links to hearing loss. Previous studies have shown that a HFD can result in microangiopathy, impaired insulin signalling, and oxidative stress in the inner ear. A HFD can also induce pathological shifts in gut microbiota and affect intestinal barrier (IB) integrity, leading to a leaky gut. A leaky gut can result in chronic systemic inflammation, which may affect extraintestinal organs. Here, we postulate that changes in gut microbiota resulting from a chronic HFD and DIO may cause a systemic inflammatory response that can compromise the permeability of the blood-labyrinth barrier (BLB) in the inner ear, thus inducing cochlear inflammation and hearing deficits.

Istradefylline Mitigates Age-Related Hearing Loss in C57BL/6J Mice.

Age-related hearing loss (ARHL) is the most common sensory disorder among older people, and yet, the treatment options are limited to medical devices such as hearing aids and cochlear implants. The high prevalence of ARHL mandates the development of treatment strategies that can prevent or rescue age-related cochlear degeneration. In this study, we investigated a novel pharmacological strategy based on inhibition of the adenosine A2A receptor (A2AR) in middle aged C57BL/6 mice prone to early onset ARHL. C57BL/6J mice were treated with weekly istradefylline (A2AR antagonist; 1 mg/kg) injections from 6 to 12 months of age. Auditory function was assessed using auditory brainstem responses (ABR) to tone pips (4-32 kHz). ABR thresholds and suprathreshold responses (wave I amplitudes and latencies) were evaluated at 6, 9, and 12 months of age. Functional outcomes were correlated with quantitative histological assessments of sensory hair cells. Cognitive function was assessed using the Morris water maze and the novel object recognition test, and the zero maze test was used to assess anxiety-like behaviour. Weekly injections of istradefylline attenuated ABR threshold shifts by approximately 20 dB at mid to high frequencies (16-32 kHz) but did not improve ABR suprathreshold responses. Istradefylline treatment improved hair cell survival in a turn-dependent manner, whilst the cognitive function was unaffected by istradefylline treatment. This study presents the first evidence for the rescue potential of istradefylline in ARHL and highlights the role of A2AR in development of age-related cochlear degeneration.

Regulator of G Protein Signalling 4 (RGS4) as a Novel Target for the Treatment of Sensorineural Hearing Loss.

We and others have previously identified signalling pathways associated with the adenosine A1 receptor (A1R) as important regulators of cellular responses to injury in the cochlea. We have shown that the "post-exposure" treatment with adenosine A1R agonists confers partial protection against acoustic trauma and other forms of sensorineural hearing loss (SNHL). The aim of this study was to determine if increasing A1R responsiveness to endogenous adenosine would have the same otoprotective effect. This was achieved by pharmacological targeting of the Regulator of G protein Signalling 4 (RGS4). RGS proteins inhibit signal transduction pathways initiated by G protein-coupled receptors (GPCR) by enhancing GPCR deactivation and receptor desensitisation. A molecular complex between RGS4 and neurabin, an intracellular scaffolding protein expressed in neural and cochlear tissues, is the key negative regulator of A1R activity in the brain. In this study, Wistar rats (6-8 weeks) were exposed to traumatic noise (110 dBSPL, 8-16 kHz) for 2 h and a small molecule RGS4 inhibitor CCG-4986 was delivered intratympanically in a Poloxamer-407 gel formulation for sustained drug release 24 or 48 h after noise exposure. Intratympanic administration of CCG-4986 48 h after noise exposure attenuated noise-induced permanent auditory threshold shifts by up to 19 dB, whilst the earlier drug administration (24 h) led to even better preservation of auditory thresholds (up to 32 dB). Significant improvement of auditory thresholds and suprathreshold responses was linked to improved survival of sensorineural tissues and afferent synapses in the cochlea. Our studies thus demonstrate that intratympanic administration of CCG-4986 can rescue cochlear injury and hearing loss induced by acoustic overexposure. This research represents a novel paradigm for the treatment of various forms of SNHL based on regulation of GPCR.

Resistance to neomycin ototoxicity in the extreme basal (hook) region of the mouse cochlea.

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.

Characterisation of cochlear inflammation in mice following acute and chronic noise exposure.

Oxidative stress has been established as the key mechanism of the cochlear damage underlying noise-induced hearing loss, however, emerging evidence suggests that cochlear inflammation may also be a major contributor. This study aimed to improve our understanding of the cochlear inflammatory response associated with acute and chronic noise exposure. C57BL/6 mice were exposed to acute traumatic noise (100 dBSPL, 8-16 kHz for 24 h) and their cochleae collected at various intervals thereafter, up to 7 days. Using quantitative RT-PCR and immunohistochemistry, changes in expression levels of proinflammatory cytokines (TNF-α, IL-1ß), chemokines (CCL2) and cell adhesion molecules (ICAM-1) were studied. All gene transcripts displayed similar dynamics of expression, with an early upregulation at 6 h post-exposure, followed by a second peak at 7 days. ICAM-1 immunoexpression increased significantly in the inferior region of the spiral ligament, peaking 24 h post-exposure. The early expression of proinflammatory mediators likely mediates the recruitment and extravasation of inflammatory cells into the noise-exposed cochlea. The occurrence of the latter expression peak is not clear, but it may be associated with reparative processes initiated in response to cochlear damage. Chronic exposure to moderate noise (90 dBSPL, 8-16 kHz, 2 h/day, up to 4 weeks) also elicited an inflammatory response, reaching a maximum after 2 weeks, suggesting that cochlear damage and hearing loss associated with chronic environmental noise exposure may be linked to inflammatory processes in the cochlea. This study thus provides further insight into the dynamics of the cochlear inflammatory response induced by exposure to acute and chronic noise.

ATP-gated ion channels mediate adaptation to elevated sound levels.

The sense of hearing is remarkable for its auditory dynamic range, which spans more than 10(12) in acoustic intensity. The mechanisms that enable the cochlea to transduce high sound levels without damage are of key interest, particularly with regard to the broad impact of industrial, military, and recreational auditory overstimulation on hearing disability. We show that ATP-gated ion channels assembled from P2X2 receptor subunits in the cochlea are necessary for the development of temporary threshold shift (TTS), evident in auditory brainstem response recordings as sound levels rise. In mice null for the P2RX2 gene (encoding the P2X2 receptor subunit), sustained 85-dB noise failed to elicit the TTS that wild-type (WT) mice developed. ATP released from the tissues of the cochlear partition with elevation of sound levels likely activates the broadly distributed P2X2 receptors on epithelial cells lining the endolymphatic compartment. This purinergic signaling is supported by significantly greater noise-induced suppression of distortion product otoacoustic emissions derived from outer hair cell transduction and decreased suprathreshold auditory brainstem response input/output gain in WT mice compared with P2RX2-null mice. At higher sound levels (≥95 dB), additional processes dominated TTS, and P2RX2-null mice were more vulnerable than WT mice to permanent hearing loss due to hair cell synapse disruption. P2RX2-null mice lacked ATP-gated conductance across the cochlear partition, including loss of ATP-gated inward current in hair cells. These data indicate that a significant component of TTS represents P2X2 receptor-dependent purinergic hearing adaptation that underpins the upper physiological range of hearing.

Mutation of the ATP-gated P2X(2) receptor leads to progressive hearing loss and increased susceptibility to noise.

Age-related hearing loss and noise-induced hearing loss are major causes of human morbidity. Here we used genetics and functional studies to show that a shared cause of these disorders may be loss of function of the ATP-gated P2X(2) receptor (ligand-gated ion channel, purinergic receptor 2) that is expressed in sensory and supporting cells of the cochlea. Genomic analysis of dominantly inherited, progressive sensorineural hearing loss DFNA41 in a six-generation kindred revealed a rare heterozygous allele, P2RX2 c.178G > T (p.V60L), at chr12:133,196,029, which cosegregated with fully penetrant hearing loss in the index family, and also appeared in a second family with the same phenotype. The mutation was absent from more than 7,000 controls. P2RX2 p.V60L abolishes two hallmark features of P2X(2) receptors: ATP-evoked inward current response and ATP-stimulated macropore permeability, measured as loss of ATP-activated FM1-43 fluorescence labeling. Coexpression of mutant and WT P2X(2) receptor subunits significantly reduced ATP-activated membrane permeability. P2RX2-null mice developed severe progressive hearing loss, and their early exposure to continuous moderate noise led to high-frequency hearing loss as young adults. Similarly, among family members heterozygous for P2RX2 p.V60L, noise exposure exacerbated high-frequency hearing loss in young adulthood. Our results suggest that P2X(2) function is required for life-long normal hearing and for protection from exposure to noise.

Properties of ATP-gated ion channels assembled from P2X2 subunits in mouse cochlear Reissner's membrane epithelial cells.

In the cochlea, Reissner's membrane separates the scala media endolymphatic compartment that sustains the positive endocochlear potential and ion composition necessary for sound transduction, from the scala vestibuli perilymphatic compartment. It is known that with sustained elevated sound levels, adenosine 5'-triphosphate (ATP) is released into the endolymph and ATP-gated ion channels on the epithelial cells lining the endolymphatic compartment shunt the electrochemical driving force, contributing to protective purinergic hearing adaptation. This study characterises the properties of epithelial cell P2X(2)-type ATP-activated membrane conductance in the mouse Reissner's membrane, which forms a substantial fraction of the scale media surface. The cells were found to express two isoforms (a and b) of the P2X(2) subunit arising from alternative splicing of the messenger RNA (mRNA) transcript that could contribute to the trimeric subunit assembly. The ATP-activated conductance demonstrated both immediate and delayed desensitisation consistent with incorporation of the combination of P2X(2) subunit isoforms. Activation by the ATP analogue 2meSATP had equipotency to ATP, whereas α,ß-meATP and adenosine 5'-diphosphate (ADP) were ineffective. Positive allosteric modulation of the P2X(2) channels by protons was profound. This native conductance was blocked by the P2X(2)-selective blocker pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) and the conductance was absent in these cells isolated from mice null for the P2rX2 gene encoding the P2X(2) receptor subunit. The activation and desensitisation properties of the Reissner's membrane epithelial cell ATP-gated P2X(2) channels likely contribute to the sensitivity and kinetics of purinergic control of the electrochemical driving force for sound transduction invoked by noise exposure.

Markers of cochlear inflammation using MRI.

PURPOSE: To quantify spatial and temporal inflammation-induced changes in vascular permeability and macrophage infiltration in guinea-pig (GP) cochlea using MRI. MATERIALS AND METHODS: GPs were injected with lipopolysaccharide (LPS) to induce cochlear inflammation. One group was injected with a gadolinium based contrast agent (GBCA) and dynamic contrast enhanced (DCE)-MRI was performed at 4, 7, and 10 days after LPS treatment. A two-compartment pharmacokinetic model was used to determine the apparent rate constant of GBCA extravasation (K(trans) ). A second group was injected with ultrasmall superparamagnetic iron oxide particles (USPIOs) and studied at 2, 3, and 7 days after LPS treatment to detect tissue USPIO uptake and correlate with histology. For both groups, control GPs were scanned similarly. RESULTS: The signal enhancement increased substantially and more rapidly at day 4 in LPS-treated than in control cochlea shortly following GBCA injection. K(trans) of LPS-treated cochlea was maximum on day 4 at 0.0218 ± 0.0032 min(-1) and then decreased to control level at 0.0036 ± 0.0004 min(-1) by day 10. In the second group, the relative signal intensity and T2 in cochlear perilymphatic spaces on day 2 decreased, on average, by 54% and 45%, respectively, compared with baseline and then remained under control levels by day 7. This suggests the infiltration of inflammatory cells, although unconfirmed by histology. CONCLUSION: This provides the first measurement of cochlear vascular permeability using MRI and a quantitative evaluation of the development of cochlear inflammation. MRI holds considerable potential for the assessment of disease processes such as clinical diagnosis of conditions such as labyrinthitis.

Expression and distribution of creatine transporter and creatine kinase (brain isoform) in developing and mature rat cochlear tissues.

Physiological processes in the cochlea associated with sound transduction and maintenance of the unique electrochemical environment are metabolically demanding. Creatine maintains ATP homeostasis by providing high-energy phosphates for ATP regeneration which is catalyzed by creatine kinase (CK). Cellular uptake of creatine requires a specific high affinity sodium- and chloride-dependent creatine transporter (CRT). This study postulates that this CRT is developmentally regulated in the rat cochlea. CRT expression was measured by quantitative real-time RT-PCR and immunohistochemistry in the postnatal (P0-P14) and adult (P22-P56) rat cochlea. The maximum CRT expression was reached at the onset of hearing (P12), and this level was maintained through to adulthood. CRT immunoreactivity was strongest in the sensory inner hair cells, supporting cells and the spiral ganglion neurons. Cochlear distribution of the CK brain isoform (CKB) was also assessed by immunohistochemistry and compared with the distribution of CRT in the developing and adult cochlea. CKB was immunolocalized in the organ of Corti supporting cells, and the lateral wall tissues involved in K(+) cycling, including stria vascularis and spiral ligament fibrocytes. Similar to CRT, CKB reached peak expression after the onset of hearing. Differential spatial and temporal expression of CRT and CK in cochlear tissues during development may reflect differential requirements for creatine-phosphocreatine buffering to replenish ATP consumed during energy-dependent metabolic processes, especially around the period when the cochlea becomes responsive to airborne sound.

The Association of Inflammatory Gut Diseases with Neuroinflammatory and Auditory Disorders.

Disorders such as inflammatory bowel disease (IBD) and celiac disease (CeD) result in intestinal hyperpermeability or 'leaky' gut. The increased permeability of the intestinal barrier allows microbial metabolites, toxins, and pathogens to infiltrate the bloodstream and extraintestinal tissues, causing systemic inflammation. Despite differences in aetiology and pathophysiology, IBD and CeD share several extraintestinal manifestations such as neuroinflammation, neurological and psychiatric manifestations, and sensorineural hearing loss (SNHL). This narrative review focuses on the association between intestinal hyperpermeability with the brain and inner ear diseases. We postulate that the microbial metabolites and pathogens released from the gut increase the permeability of natural barriers, such as the blood-brain barrier (BBB) and blood-labyrinth barrier (BLB). The barrier breakdown allows the spreading of inflammatory processes to the brain and inner ear, leading to disease.