Adenosine A1 Receptor Protects Against Cisplatin Ototoxicity by Suppressing the NOX3/STAT1 Inflammatory Pathway in the Cochlea.

Cisplatin is a commonly used antineoplastic agent that produces ototoxicity that is mediated in part by increasing levels of reactive oxygen species (ROS) via the NOX3 NADPH oxidase pathway in the cochlea. Recent studies implicate ROS generation in mediating inflammatory and apoptotic processes and hearing loss by activating signal transducer and activator of transcription (STAT1). In this study, we show that the adenosine A1 receptor (A1AR) protects against cisplatin ototoxicity by suppressing an inflammatory response initiated by ROS generation via NOX3 NADPH oxidase, leading to inhibition of STAT1. Trans-tympanic administration of the A1AR agonist R-phenylisopropyladenosine (R-PIA) inhibited cisplatin-induced ototoxicity, as measured by auditory brainstem responses and scanning electron microscopy in male Wistar rats. This was associated with reduced NOX3 expression, STAT1 activation, tumor necrosis factor-α (TNF-α) levels, and apoptosis in the cochlea. In vitro studies in UB/OC-1 cells, an organ of Corti immortalized cell line, showed that R-PIA reduced cisplatin-induced phosphorylation of STAT1 Ser(727) (but not Tyr(701)) and STAT1 luciferase activity by suppressing the ERK1/2, p38, and JNK mitogen-activated protein kinase (MAPK) pathways.R-PIA also decreased the expression of STAT1 target genes, such as TNF-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) and reduced cisplatin-mediated apoptosis. These data suggest that the A1AR provides otoprotection by suppressing NOX3 and inflammation in the cochlea and could serve as an ideal target for otoprotective drug therapy. SIGNIFICANCE STATEMENT: Cisplatin is a widely used chemotherapeutic agent for the treatment of solid tumors. Its use results in significant and permanent hearing loss, for which no US Food and Drug Administration-approved treatment is currently available. In this study, we targeted the cochlear adenosine A1 receptor (A1AR) by trans-tympanic injections of the agonist R-phenylisopropyladenosine (R-PIA) and showed that it reduced cisplatin-induced inflammation and apoptosis in the rat cochlea and preserved hearing. The mechanism of protection involves suppression of the NOX3 NADPH oxidase enzyme, a major target of cisplatin-induced reactive oxygen species (ROS) generation in the cochlea. ROS initiates an inflammatory and apoptotic cascade in the cochlea by activating STAT1 transcription factor, which is attenuated byR-PIA. Therefore, trans-tympanic delivery of A1AR agonists could effectively treat cisplatin ototoxicity.

Adenosine receptors: expression, function and regulation.

Adenosine receptors (ARs) comprise a group of G protein-coupled receptors (GPCR) which mediate the physiological actions of adenosine. To date, four AR subtypes have been cloned and identified in different tissues. These receptors have distinct localization, signal transduction pathways and different means of regulation upon exposure to agonists. This review will describe the biochemical characteristics and signaling cascade associated with each receptor and provide insight into how these receptors are regulated in response to agonists. A key property of some of these receptors is their ability to serve as sensors of cellular oxidative stress, which is transmitted by transcription factors, such as nuclear factor (NF)-κB, to regulate the expression of ARs. Recent observations of oligomerization of these receptors into homo- and heterodimers will be discussed. In addition, the importance of these receptors in the regulation of normal and pathological processes such as sleep, the development of cancers and in protection against hearing loss will be examined.

siRNA-mediated knock-down of NOX3: therapy for hearing loss?

Cisplatin is a widely used chemotherapeutic agent that causes significant hearing loss. Previous studies have shown that cisplatin exposure is associated with increase in reactive oxygen species (ROS) in the cochlea. The inner ear expresses a unique isoform of NADPH oxidase, NOX3. This enzyme may be the primary source of ROS generation in the cochlea. The knockdown of NOX3 by pretreatment with siRNA prevented cisplatin ototoxicity, as demonstrated by preservation of hearing thresholds and inner ear sensory cells. Trans-tympanic NOX3 siRNA reduced the expression of NOX3 and biomarkers of cochlear damage, including transient receptor vanilloid 1 (TRPV1) channel and kidney injury molecule-1 (KIM-1) in cochlear tissues. In addition, siRNA against NOX3 reduced apoptosis as demonstrated by TUNEL staining, and prevented the increased expression of Bax and abrogated the decrease in Bcl2 expression following cisplatin administration. Trans-tympanic administration of siRNA directed against NOX3 may provide a useful method of attenuating cisplatin ototoxicity. In this paper, we review recent publications dealing with the role of NOX3 in ototoxicity and the effects of siRNA against cisplatin-induced hearing loss.

Effects of natural products on cisplatin ototoxicity and chemotherapeutic efficacy.

INTRODUCTION: Cisplatin is a very effective chemotherapeutic agent against a variety of solid tumors. Unfortunately, cisplatin causes permanent sensorineural hearing loss in at least two-thirds of patients treated. There are no FDA approved drugs to prevent this serious side effect. AREAS COVERED: This paper reviews various natural products that ameliorate cisplatin ototoxicity. These compounds are strong antioxidants and anti-inflammatory agents. This review includes mostly preclinical studies but also discusses a few small clinical trials with natural products to minimize hearing loss from cisplatin chemotherapy in patients. The interactions of natural products with cisplatin in tumor-bearing animal models are highlighted. A number of natural products did not interfere with cisplatin anti-tumor efficacy and some agents actually potentiated cisplatin anti-tumor activity. EXPERT OPINION: There are a number of natural products or their derivatives that show excellent protection against cisplatin ototoxicity in preclinical studies. There is a need to insure uniform standards for purity of drugs derived from natural sources and to ensure adequate pharmacokinetics and safety of these products. Natural products that protect against cisplatin ototoxicity and augment cisplatin's anti-tumor effects in multiple studies of tumor-bearing animals are most promising for advancement to clinical trials. The most promising natural products include honokiol, sulforaphane, and thymoquinone.

Targeting CXCL1 chemokine signaling for treating cisplatin ototoxicity.

Cisplatin is chemotherapy used for solid tumor treatment like lung, bladder, head and neck, ovarian and testicular cancers. However, cisplatin-induced ototoxicity limits the utility of this agent in cancer patients, especially when dose escalations are needed. Ototoxicity is associated with cochlear cell death through DNA damage, the generation of reactive oxygen species (ROS) and the consequent activation of caspase, glutamate excitotoxicity, inflammation, apoptosis and/or necrosis. Previous studies have demonstrated a role of CXC chemokines in cisplatin ototoxicity. In this study, we investigated the role of CXCL1, a cytokine which increased in the serum and cochlea by 24 h following cisplatin administration. Adult male Wistar rats treated with cisplatin demonstrated significant hearing loss, assessed by auditory brainstem responses (ABRs), hair cell loss and loss of ribbon synapse. Immunohistochemical studies evaluated the levels of CXCL1 along with increased presence of CD68 and CD45-positive immune cells in cochlea. Increases in CXCL1 was time-dependent in the spiral ganglion neurons and organ of Corti and was associated with progressive increases in CD45, CD68 and IBA1-positive immune cells. Trans-tympanic administration of SB225002, a chemical inhibitor of CXCR2 (receptor target for CXCL1) reduced immune cell migration, protected against cisplatin-induced hearing loss and preserved hair cell integrity. We show that SB225002 reduced the expression of CXCL1, NOX3, iNOS, TNF-α, IL-6 and COX-2. Similarly, knockdown of CXCR2 by trans-tympanic administration of CXCR2 siRNA protected against hearing loss and loss of outer hair cells and reduced ribbon synapses. In addition, SB225002 reduced the expression of inflammatory mediators induced by cisplatin. These results implicate the CXCL1 chemokine as an early player in cisplatin ototoxicity, possibly by initiating the immune cascade, and indicate that CXCR2 is a relevant target for treating cisplatin ototoxicity.

Transient Receptor Potential Channels and Auditory Functions.

Significance: Transient receptor potential (TRP) channels are cation-gated channels that serve as detectors of various sensory modalities, such as pain, heat, cold, and taste. These channels are expressed in the inner ear, suggesting that they could also contribute to the perception of sound. This review provides more details on the different types of TRP channels that have been identified in the cochlea to date, focusing on their cochlear distribution, regulation, and potential contributions to auditory functions. Recent Advances: To date, the effect of TRP channels on normal cochlear physiology in mammals is still unclear. These channels contribute, to a limited extent, to normal cochlear physiology such as the hair cell mechanoelectrical transduction channel and strial functions. More detailed information on a number of these channels in the cochlea awaits future studies. Several laboratories focusing on TRPV1 channels have shown that they are responsive to cochlear stressors, such as ototoxic drugs and noise, and regulate cytoprotective and/or cell death pathways. TRPV1 expression in the cochlea is under control of oxidative stress (produced primarily by NOX3 NADPH oxidase) as well as STAT1 and STAT3 transcription factors, which differentially modulate inflammatory and apoptotic signals in the cochlea. Inhibition of oxidative stress or inflammation reduces the expression of TRPV1 channels and protects against cochlear damage and hearing loss. Critical Issues: TRPV1 channels are activated by both capsaicin and cisplatin, which produce differential effects on the inner ear. How these differential actions are produced is yet to be determined. It is clear that TRPV1 is an essential component of cisplatin ototoxicity as knockdown of these channels protects against hearing loss. In contrast, activation of TRPV1 by capsaicin protected against subsequent hearing loss induced by cisplatin. The cellular targets that are influenced by these two drugs to account for their differential profiles need to be fully elucidated. Furthermore, the potential involvement of different TRP channels present in the cochlea in regulating cisplatin ototoxicity needs to be determined. Future Directions: TRPV1 has been shown to mediate the entry of aminoglycosides into the hair cells. Thus, novel otoprotective strategies could involve designing drugs to inhibit entry of aminoglycosides and possibly other ototoxins into cochlear hair cells. TRP channels, including TRPV1, are expressed on circulating and resident immune cells. These receptors modulate immune cell functions. However, whether they are activated by cochlear stressors to initiate cochlear inflammation and ototoxicity needs to be determined. A better understanding of the function and regulation of these TRP channels in the cochlea could enable development of novel treatments for treating hearing loss. Antioxid. Redox Signal. 36, 1158-1170.

Lmo4 Deficiency Enhances Susceptibility to Cisplatin-Induced Cochlear Apoptosis and Hearing Loss.

Cisplatin, a potent chemotherapeutic drug, induces ototoxicity, which limits its clinical utility. Cisplatin-induced oxidative stress plays a causal role in cochlear apoptosis while the consequent nitrative stress leads to the nitration of LIM domain only 4 (LMO4), a transcriptional regulator, and decreases its cochlear expression levels. Here, we show a direct link between cochlear LMO4 and cisplatin-induced hearing loss by employing a Lmo4 conditional knockout mouse model (Lmo4lox/lox; Gfi1Cre/+). Hair cell-specific deletion of Lmo4 did not alter cochlear morphology or affect hearing thresholds and otoacoustic emissions, in the absence of apoptotic stimuli. Cisplatin treatment significantly elevated the auditory brainstem response thresholds of conditional knockouts, across all frequencies. Moreover, deletion of Lmo4 compromised the activation of STAT3, a downstream target that regulates anti-apoptotic machinery. Immunostaining indicated that the expression of phosphorylated STAT3 was significantly decreased while the expression of activated caspase 3 was significantly increased in Lmo4 deficient hair cells, post-cisplatin treatment. These findings suggest an otoprotective role of LMO4 as cisplatin-induced decrease in cochlear LMO4 could compromise the LMO4/STAT3 cellular defense mechanism to induce ototoxicity.

Ototoxicity of Non-aminoglycoside Antibiotics.

It is well-known that aminoglycoside antibiotics can cause significant hearing loss and vestibular deficits that have been described in animal studies and in clinical reports. The purpose of this review is to summarize relevant preclinical and clinical publications that discuss the ototoxicity of non-aminoglycoside antibiotics. The major classes of antibiotics other than aminoglycosides that have been associated with hearing loss in animal studies and in patients are discussed in this report. These antibiotics include: capreomycin, a polypeptide antibiotic that has been used to treat patients with drug-resistant tuberculosis, particularly in developing nations; the macrolides, including erythromycin, azithromycin and clarithromycin; and vancomycin. These antibiotics have been associated with ototoxicity, particularly in neonates. It is critical to be aware of the ototoxic potential of these antibiotics since so much attention has been given to the ototoxicity of aminoglycoside antibiotics in the literature.

Oxidative Stress and Inflammation Caused by Cisplatin Ototoxicity.

Hearing loss is a significant health problem that can result from a variety of exogenous insults that generate oxidative stress and inflammation. This can produce cellular damage and impairment of hearing. Radiation damage, ageing, damage produced by cochlear implantation, acoustic trauma and ototoxic drug exposure can all generate reactive oxygen species in the inner ear with loss of sensory cells and hearing loss. Cisplatin ototoxicity is one of the major causes of hearing loss in children and adults. This review will address cisplatin ototoxicity. It includes discussion of the mechanisms associated with cisplatin-induced hearing loss including uptake pathways for cisplatin entry, oxidative stress due to overpowering antioxidant defense mechanisms, and the recently described toxic pathways that are activated by cisplatin, including necroptosis and ferroptosis. The cochlea contains G-protein coupled receptors that can be activated to provide protection. These include adenosine A1 receptors, cannabinoid 2 receptors (CB2) and the Sphingosine 1-Phosphate Receptor 2 (S1PR2). A variety of heat shock proteins (HSPs) can be up-regulated in the cochlea. The use of exosomes offers a novel method of delivery of HSPs to provide protection. A reversible MET channel blocker that can be administered orally may block cisplatin uptake into the cochlear cells. Several protective agents in preclinical studies have been shown to not interfere with cisplatin efficacy. Statins have shown efficacy in reducing cisplatin ototoxicity without compromising patient response to treatment. Additional clinical trials could provide exciting findings in the prevention of cisplatin ototoxicity.

Regulator of G protein signaling 17 represents a novel target for treating cisplatin induced hearing loss.

Regulators of G protein signaling (RGS) accelerate the GTPase activity of G proteins to enable rapid termination of the signals triggered by G protein-coupled receptors (GPCRs). Activation of several GPCRs, including cannabinoid receptor 2 (CB2R) and adenosine A1 receptor (A1AR), protects against noise and drug-induced ototoxicity. One such drug, cisplatin, an anticancer agent used to treat various solid tumors, produces permanent hearing loss in experimental animals and in a high percentage of cancer patients who undergo treatments. In this study we show that cisplatin induces the expression of the RGS17 gene and increases the levels of RGS17 protein which contributes to a significant proportion of the hearing loss. Knockdown of RGS17 suppressed cisplatin-induced hearing loss in male Wistar rats, while overexpression of RGS17 alone produced hearing loss in vivo. Furthermore, RGS17 and CB2R negatively regulate the expression of each other. These data suggest that RGS17 mediates cisplatin ototoxicity by uncoupling cytoprotective GPCRs from their normal G protein interactions, thereby mitigating the otoprotective contributions of endogenous ligands of these receptors. Thus, RGS17 represents a novel mediator of cisplatin ototoxicity and a potential therapeutic target for treating hearing loss.

Role of beta-arrestin1/ERK MAP kinase pathway in regulating adenosine A1 receptor desensitization and recovery.

Exposure of cells to adenosine receptor (AR) agonists leads to receptor uncoupling from G proteins and downregulation of the A(1)AR. The receptor levels on the cell surface generally recover on withdrawal of the agonist, because of either translocation of the sequestered A(1)AR back to plasma membrane or de novo synthesis of A(1)AR. To examine the mechanism(s) underlying A(1)AR downregulation and recovery, we treated ductus deferens tumor (DDT(1) MF-2) cells with the agonist R-phenylisopropyladenosine (R-PIA) and showed a decrease in membrane A(1)AR levels by 24 h, which was associated with an unexpected 11-fold increase in A(1)AR mRNA. Acute exposure of these cells to R-PIA resulted in a rapid translocation of beta-arrestin1 to the plasma membrane. Knockdown of beta-arrestin1 by short interfering RNA (siRNA) blocked R-PIA-mediated downregulation of the A(1)AR, suppressed R-PIA-dependent ERK1/2 and activator protein-1 (AP-1) activity, and reduced the induction of A(1)AR mRNA. Withdrawal of the agonist after a 24-h exposure resulted in rapid recovery of plasma membrane A(1)AR. This was dependent on the de novo protein synthesis and on the activity of ERK1/2 but independent of beta-arrestin1 and nuclear factor-kappaB. Together, these data suggest that exposure to A(1)AR agonist stimulates ERK1/2 activity via beta-arrestin1, which subserves receptor uncoupling and downregulation, in addition to the induction of A(1)AR expression. We propose that such a pathway ensures both the termination of the agonist signal and recovery by priming the cell for rapid de novo synthesis of A(1)AR once the drug is terminated.

Strategies to reduce the risk of platinum containing antineoplastic drug-induced ototoxicity.

INTRODUCTION: Cisplatin is a highly effective chemotherapeutic agent against a variety of solid tumors in adults and in children. Unfortunately, a large percentage of patients suffer permanent sensorineural hearing loss. Up to 60% of children and at least 50% of adults suffer this complication that seriously compromises their quality of life. Hearing loss is due to damage to the sensory cells in the inner ear. The mechanisms of cochlear damage are still being investigated. However, it appears that inner ear damage is triggered by reactive oxygen species (ROS) formation and inflammation 34. AREAS COVERED: We discuss a number of potential therapeutic targets that can be addressed to provide hearing protection. These strategies include enhancing the endogenous antioxidant pathways, heat shock proteins, G protein coupled receptors and counteracting ROS and reactive nitrogen species, and blocking pathways that produce inflammation, including TRPV1 and STAT1 36. EXPERT OPINION: Numerous potential protective agents show promise in animal models by systemic or local administration. However, clinical trials have not shown much efficacy to date with the exception of sodium thiosulfate. There is an urgent need to discover safe and effective protective agents that do not interfere with the efficacy of cisplatin against tumors yet preserve hearing 151.

Short interfering RNA against transient receptor potential vanilloid 1 attenuates cisplatin-induced hearing loss in the rat.

Cisplatin, a chemotherapeutic agent of choice for the treatment of solid tumors, produces hearing loss in approximately half a million new cancer patients annually in the United States. The hearing loss is due, in part, to increased generation of reactive oxygen species (ROS) in the cochlea, leading to lipid peroxidation and damage or death of outer hair cells in the organ of Corti. The cochlea expresses the transient receptor potential vanilloid 1 (TRPV1), which are normally expressed on small diameter neurons in the peripheral nervous system and mediate thermal sensitivity, but whose role in the cochlea is unclear. In this study, we show that TRPV1 is coregulated along with the NADPH oxidase isoform, NOX3, by cisplatin. Induction of these proteins by cisplatin is dependent on ROS generation, since it is reversed by systemic lipoic acid administration. In organ of Corti hair cell cultures (UB/OC-1 cells), cisplatin activates and induces TRPV1 and NOX3, leading to apoptosis of these cells. Inhibition of TRPV1 by capsazepine or ruthenium red reduced the apoptosis, implicating TRPV1 in this process. Treatment of UB/OC-1 cultures with short interfering RNA (siRNA) against either TRPV1 or NOX3 reduced cisplatin-induced apoptosis, while round window application of TRPV1 siRNA to rats reduced TRPV1 expression, decreased damage to outer hair cells and reduced cisplatin-induced hearing loss. These data provide a link between NOX3 and TRPV1 in cisplatin-induced hearing loss and suggest that targeting these proteins for knockdown by siRNA could serve as a novel approach in treating cisplatin ototoxicity.

Cisplatin ototoxicity and protection: clinical and experimental studies.

Cisplatin is a chemotherapeutic agent that is widely used to treat a variety of malignant tumors. Serious dose-limiting side effects like ototoxicity, nephrotoxicity and neurotoxicity occur with the use of this agent. This review summarizes recent important clinical and experimental investigations of cisplatin ototoxicity. It also discusses the utility of protective agents employed in patients and in experimental animals. The future strategies for limiting cisplatin ototoxicity will need to avoid interference with the therapeutic effect of cisplatin in order to enhance the quality of life of patients receiving this important anti-tumor agent.

Mechanisms of Cisplatin-Induced Ototoxicity and Prevention.

Cisplatin is a highly effective antineoplastic agent used to treat solid tumors. Unfortunately, the administration of this drug leads to significant side effects, including ototoxicity, nephrotoxicity, and neurotoxicity. This review addresses the mechanisms of cisplatin-induced ototoxicity and various strategies tested to prevent this distressing adverse effect. The molecular pathways underlying cisplatin ototoxicity are still being investigated. Cisplatin enters targeted cells in the cochlea through the action of several transporters. Once it enters the cochlea, cisplatin is retained for months to years. It can cause DNA damage, inhibit protein synthesis, and generate reactive oxygen species that can lead to inflammation and apoptosis of outer hair cells, resulting in permanent hearing loss. Strategies to prevent cisplatin ototoxicity have utilized antioxidants, transport inhibitors, G-protein receptor agonists, and anti-inflammatory agents. There are no FDA-approved drugs to prevent cisplatin ototoxicity. It is critical that potential protective agents do not interfere with the antitumor efficacy of cisplatin.

Local Drug Delivery for Prevention of Hearing Loss.

Systemic delivery of therapeutics for targeting the cochlea to prevent or treat hearing loss is challenging. Systemic drugs have to cross the blood-labyrinth barrier (BLB). BLB can significantly prevent effective penetration of drugs in appropriate concentrations to protect against hearing loss caused by inflammation, ototoxic drugs, or acoustic trauma. This obstacle may be obviated by local administration of protective agents. This route can deliver higher concentration of drug compared to systemic application and preclude systemic side effects. Protective agents have been administered by intra-tympanic injection in numerous preclinical studies. Drugs such as steroids, etanercept, D and L-methionine, pifithrin-alpha, adenosine agonists, melatonin, kenpaullone (a cyclin-dependent kinase 2 (CDK2) inhibitor) have been reported to show efficacy against cisplatin ototoxicity in animal models. Several siRNAs have been shown to ameliorate cisplatin ototoxicity when administered by intra-tympanic injection. The application of corticosteroids and a number of other drugs with adjuvants appears to enhance efficacy. Administration of siRNAs to knock down AMPK kinase, liver kinase B1 (LKB1) or G9a in the cochlea have been found to ameliorate noise-induced hearing loss. The local administration of these compounds appears to be effective in protecting the cochlea against damage from cisplatin or noise trauma. Furthermore the intra-tympanic route yields maximum protection in the basal turn of the cochlea which is most vulnerable to cisplatin ototoxicity and noise trauma. There appears to be very little transfer of these agents to the systemic circulation. This would avoid potential side effects including interference with anti-tumor efficacy of cisplatin. Nanotechnology offers strategies to effectively deliver protective agents to the cochlea. This review summarizes the pharmacology of local drug delivery by intra-tympanic injection to prevent hearing loss caused by cisplatin and noise exposure in animals. Future refinements in local protective agents provide exciting prospects for amelioration of hearing loss resulting from cisplatin or noise exposure.

Oral Administration of Caffeine Exacerbates Cisplatin-Induced Hearing Loss.

Adenosine A1 receptors (A1AR) are well characterized for their role in cytoprotection. Previous studies have demonstrated the presence of these receptors in the cochlea where their activation were shown to suppress cisplatin-induced inflammatory response and the resulting ototoxicity. Inhibition of A1AR by caffeine, a widely consumed psychoactive substance, could antagonize the endogenous protective role of these receptors in cochlea and potentiate cisplatin-induced hearing loss. This hypothesis was tested in a rat model of cisplatin ototoxicity following oral administration of caffeine. We report here that single-dose administration of caffeine exacerbates cisplatin-induced hearing loss without increasing the damage to outer hair cells (OHCs), but increased synaptopathy and inflammation in the cochlea. These effects of caffeine were mediated by its blockade of A1AR, as co-administration of R-PIA, an A1AR agonist, reversed the detrimental actions of caffeine and cisplatin on hearing loss. Multiple doses of caffeine exacerbated cisplatin ototoxicity which was associated with damage to OHCs and cochlear synaptopathy. These findings highlight a possible drug-drug interaction between caffeine and cisplatin for ototoxicity and suggest that caffeine consumption should be cautioned in cancer patients treated with a chemotherapeutic regimen containing cisplatin.

Targeting Inflammatory Processes Mediated by TRPVI and TNF-α for Treating Noise-Induced Hearing Loss.

Noise trauma is the most common cause of hearing loss in adults. There are no known FDA approved drugs for prevention or rescue of noise-induced hearing loss (NIHL). In this study, we provide evidence that implicates stress signaling molecules (TRPV1, NOX3, and TNF-α) in NIHL. Furthermore, we provide evidence that inhibiting any one of these moieties can prevent and treat NIHL when administered within a window period. Hearing loss induced by loud noise is associated with the generation of reactive oxygen species (ROS), increased calcium (Ca2+) in the endolymph and hair cells, and increased inflammation in the cochlea. Increased (Ca2+) and ROS activity persists for several days after traumatic noise exposure (NE). Chronic increases in (Ca2+) and ROS have been shown to increase inflammation and apoptosis in various tissue. However, the precise role of Ca2+ up-regulation and the resulting inflammation causing a positive feedback loop in the noise-exposed cochlea to generate sustained toxic amounts of Ca2+ are unknown. Here we show cochlear TRPV1 dysregulation is a key step in NIHL, and that inflammatory TNF-α cytokine-mediated potentiation of TRPV1 induced Ca2+ entry is an essential mechanism of NIHL. In the Wistar rat model, noise produces an acute (within 48 h) and a chronic (within 21 days) increase in cochlear gene expression of TRPV1, NADPH oxidase 3 (NOX3) and pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX2). Additionally, we also show that H2O2 (100 µM) produces a robust increase in Ca2+ entry in cell cultures which is enhanced by TNF-α via the TRPV1 channel and which involves ERK1/2 phosphorylation. Mitigation of NIHL could be achieved by using capsaicin (TRPV1 agonist that rapidly desensitizes TRPV1. This mechanism is used in the treatment of pain in diabetic peripheral neuropathy) pretreatment or by inhibition of TNF-α with Etanercept (ETA), administered up to 7 days prior to NE or within 24 h of noise. Our results demonstrate the importance of the synergistic interaction between TNF-α and TRPV1 in the cochlea and suggest that these are important therapeutic targets for treating NIHL.

Capsaicin Protects Against Cisplatin Ototoxicity by Changing the STAT3/STAT1 Ratio and Activating Cannabinoid (CB2) Receptors in the Cochlea.

Capsaicin, the spicy component of hot chili peppers activates the TRPV1 pain receptors, and causes rapid desensitization. Capsaicin also ameliorates cisplatin-induced nephrotoxicity. Cisplatin, a commonly used anti-neoplastic agent for solid tumors causes significant hearing loss, nephrotoxicity and peripheral neuropathy. Upregulation of cochlear TRPV1 expression is related to cisplatin-mediated ototoxicity. Here we report that direct TRPV1 activation by localized trans-tympanic (TT) or oral administration of capsaicin (TRPV1 agonist) prevents cisplatin ototoxicity by sustained increased activation of pro-survival transcription factor signal transducer and activator of transcription (STAT3) in the Wistar rat. Cisplatin treatment produced prolonged activation of pro-apoptotic Ser727 p-STAT1 and suppressed Tyr705-p-STAT3 for up to 72 h in the rat cochlea. Our data indicate that capsaicin causes a transient STAT1 activation via TRPV1 activation, responsible for the previously reported temporary threshold shift. Additionally, we found that capsaicin increased cannabinoid receptor (CB2) in the cochlea, which leads to pro-survival Tyr705-p-STAT3 activation. This tilts the delicate balance of p-STAT3/p-STAT1 towards survival. Furthermore, capsaicin mediated protection is lost when CB2 antagonist AM630 is administered prior to capsaicin treatment. In conclusion, capsaicin otoprotection appears to be mediated by activation of CB2 receptors in the cochlea which are coupled to both STAT1 and STAT3 activation.

Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity.

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.