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Abstract Transforming growth factor-β (TGF-β) signaling plays a significant role in multiple biological processes, including inflammation, immunity, and cell death. However, its specific impact on the cochlea remains unclear. In this study, we aimed to investigate the effects of TGF-β signaling suppression on auditory function and cochlear pathology in mice with kanamycin-induced ototoxicity. Kanamycin and furosemide (KM-FS) were systemically administered to 8-week-old C57/BL6 mice, followed by immediate topical application of a TGF-β receptor inhibitor (TGF-βRI) onto the round window membrane. Results showed significant TGF-β receptor upregulation in spiral ganglion neurons (SGNs) after KM-FA ototoxicity, whereas expression levels in the TGF-βRI treated group remained unchanged. Interestingly, despite no significant change in cochlear TGF-β expression after KM-FS ototoxicity, TGF-βRI treatment resulted in a significant decrease in TGF-β signaling. Regarding auditory function, TGF-βRI treatment offered no therapeutic effects on hearing thresholds and hair cell survival following KM-FS ototoxicity. However, SGN loss and macrophage infiltration were significantly increased with TGF-βRI treatment. These results imply that inhibition of TGF-β signaling after KM-FS ototoxicity promotes cochlear inflammation and SGN degeneration.

Transforming growth factor-β (TGF-β) signaling plays a significant role in multiple biological processes, including inflammation, immunity, and cell death. However, its specific impact on the cochlea remains unclear. In this study, we aimed to investigate the effects of TGF-β signaling suppression on auditory function and cochlear pathology in mice with kanamycin-induced ototoxicity. Kanamycin and furosemide (KM-FS) were systemically administered to 8-week-old C57/BL6 mice, followed by immediate topical application of a TGF-β receptor inhibitor (TGF-βRI) onto the round window membrane. Results showed significant TGF-β receptor upregulation in spiral ganglion neurons (SGNs) after KM-FA ototoxicity, whereas expression levels in the TGF-βRI treated group remained unchanged. Interestingly, despite no significant change in cochlear TGF-β expression after KM-FS ototoxicity, TGF-βRI treatment resulted in a significant decrease in TGF-β signaling. Regarding auditory function, TGF-βRI treatment offered no therapeutic effects on hearing thresholds and hair cell survival following KM-FS ototoxicity. However, SGN loss and macrophage infiltration were significantly increased with TGF-βRI treatment. These results imply that inhibition of TGF-β signaling after KM-FS ototoxicity promotes cochlear inflammation and SGN degeneration. [ABSTRACT FROM AUTHOR]

Radiotherapy is a crucial treatment for head and neck malignancies, but it can sometimes cause sensorineural hearing loss (SNHL). Changes in the immune microenvironment and sensory neuroepithelium of the inner ear after radiation exposure remain poorly understood. This study investigated cochlear morphology and macrophages in the inner ear after high-dose irradiation. The heads of heterozygous 8-week-old Cx3cr1GFP/+ male mice were irradiated with 30Gy of X-rays and biological samples were collected on days 1, 7, and 10 after irradiation. Auditory brainstem responses were used to assess auditory function in the mice. Changes in basilar membrane hair cells, spiral ganglion neurons (SGN), and inner ear macrophages were observed using hematoxylin-eosin (HE) staining and immunofluorescence staining. The expression of inflammatory mediators in the inner ear was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) in cochlear tissue. The results showed no significant hair cell loss after a single high dose of radiation. However, the mice developed pantonal hearing loss on day 10 when HE staining revealed SGN atrophy and immunofluorescence showed decreased neurofilament expression. The number of macrophages in the inner ear reduced over time. RT-qPCR showed that cochlear inflammatory factors and chemokines were briefly upregulated on day 1st after irradiation and then decreased over time. In conclusion, high-dose irradiation causes acute SNHL that is not associated with hair cell loss and may be related to SGN changes. Radiation-induced SNHL is associated with a reduction in cochlear macrophages and changes in the immune microenvironment, but the relationship between the two remains to be investigated.

AIM: To study whether glycyrrhizic acid (GL) can resist the ototoxicity of cisplatin( CDDP) in mice and its molecular mechanism. METHODS： Male C57BL/6J mice were divided into 5 groups： control group, DMSO(5%) group, CDDP(4 mg/kg) group, CDDP+low-dose(50 mg/kg) GL group, and CDDP+high-dose(100 mg/kg) GL group(n=14) . Auditory brainstem response(ABR) was used to detect hearing changes of mice. HE staining was used to observe the morphological change of cochlear stria vascular in mice. Evans blue(EB) staining was used to observe the per meability change of the blood-labyrinth barrier (BLB) . Immunohistochemical technique was used to detect the expression and distribution of adhesion protein VE-cadherin and tight junction protein ZO-1 on the cochlear stria. ELISA assay and immunofluorescence technology were employed to detect the expression of tumor necrosis factor-α( TNF-α) and interleu kin-1β( 1L-1β) . RESULTS： In CDDP group, ABR waveforms of all frequencies were disturbed, the hearing threshold was significantly increased, and I wave latency was prolonged (P<0. 05) . In CDDP+GL group, ABR waveforms of various frequencies were well differentiated, the hearing threshold was significantly decreased, and the latency of I-wave was shortened(P<0. 01) . The disordered morphology and more vacuoles in the stria vascularis were observed by HE staining in CDDP group. The GL alleviated CDDP-induced damage in the stria vascularis. In EB staining, CDDP caused an increase in per meability of BLB(P<0. 01), which was improved by GL treatment (P<0. 01) . Immunohistochemical results showed that the expression of VE-cadherin and ZO-1 in CDDP group were decreased (P<0. 01), which was restored in CDDP+GL group(P<0. 01) . The ELISA and immunofluorescence results showed that the expression of IL-1β and TNF-α was increased after CDDP treatment(P<0. 01), which was restored in CDDP+GL group(P<0. 01) . CONCLUSION: The GL alleviates CDDP-induced hearing loss in mice by inhibiting CDDP-induced inflammation and reducing the permeability of BLB. [ABSTRACT FROM AUTHOR]

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

Sudden sensorineural hearing loss (SSNHL) is routinely encountered and is one of the most common emergent diseases in otolaryngology clinics. However, the etiology of SSNHL remains unclear. Due to the inaccessibility of the living human inner ear for biopsy, studies investigating the etiology of SSNHL have been performed by analyzing data obtained from examinations using peripheral blood or imaging. We updated the findings obtained from serological, magnetic resonance imaging, genetic, and viral examinations to reveal the etiology of SSNHL. Regarding viral examination, we focused on sensorineural hearing loss associated with coronavirus disease (COVID-19) because the number of correlated reports has been increasing after the outbreak. The updated findings revealed the following three possible mechanisms underlying the development of SSNHL: thrombosis and resulting vascular obstruction in the cochlea, asymptomatic viral infection and resulting damage to the cochlea, and cochlear inflammation and resulting damage to the cochlea. Thrombosis and viral infection are predominant, and cochlear inflammation can be secondarily induced through viral infection or even thrombosis. The findings about sensorineural hearing loss associated with COVID-19 supported the possibility that asymptomatic viral infection is one of the etiologies of SSNHL, and the virus can infect inner ear tissues and directly damage them. [ABSTRACT FROM AUTHOR]

Radiotherapy is a crucial treatment for head and neck malignancies, but it can sometimes cause sensorineural hearing loss (SNHL). Changes in the immune microenvironment and sensory neuroepithelium of the inner ear after radiation exposure remain poorly understood. This study investigated cochlear morphology and macrophages in the inner ear after high-dose irradiation. The heads of heterozygous 8-week-old Cx3cr1 GFP/+ male mice were irradiated with 30Gy of X-rays and biological samples were collected on days 1, 7, and 10 after irradiation. Auditory brainstem responses were used to assess auditory function in the mice. Changes in basilar membrane hair cells, spiral ganglion neurons (SGN), and inner ear macrophages were observed using hematoxylin-eosin (HE) staining and immunofluorescence staining. The expression of inflammatory mediators in the inner ear was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) in cochlear tissue. The results showed no significant hair cell loss after a single high dose of radiation. However, the mice developed pantonal hearing loss on day 10 when HE staining revealed SGN atrophy and immunofluorescence showed decreased neurofilament expression. The number of macrophages in the inner ear reduced over time. RT-qPCR showed that cochlear inflammatory factors and chemokines were briefly upregulated on day 1st after irradiation and then decreased over time. In conclusion, high-dose irradiation causes acute SNHL that is not associated with hair cell loss and may be related to SGN changes. Radiation-induced SNHL is associated with a reduction in cochlear macrophages and changes in the immune microenvironment, but the relationship between the two remains to be investigated., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 Published by Elsevier Ltd.)

Summary: Background: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss. Methods: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context. Findings: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice. Interpretation: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss. Funding: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine.

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. [ABSTRACT FROM AUTHOR]

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.

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators.

Drug- and noise-related hearing loss are both associated with inflammatory responses in the inner ear. We propose that intracochlear delivery of a combination of pro-resolving mediators, specialized proteins and lipids that accelerate the return to homeostasis by modifying the immune response rather than by inhibiting inflammation, might have a profound effect on the prevention of sensorineural hearing loss. However, intracochlear delivery of such agents requires a reliable and effective method to convey them, fully active, directly to the target cells. The present study provides evidence that extracellular vesicles (EVs) from auditory HEI-OC1 cells may incorporate significant quantities of anti-inflammatory drugs, pro-resolving mediators and their polyunsaturated fatty acid precursors as cargo, and potentially could work as carriers for their intracochlear delivery. EVs generated by HEI-OC1 cells were divided by size into two fractions, small (≤150 nm diameter) and large (>150 nm diameter), and loaded with aspirin, lipoxin A4, resolvin D1, and the polyunsaturated fatty acids (PUFA) arachidonic, eicosapentaenoic, docosahexanoic, and linoleic. Bottom-up proteomics revealed a differential distribution of selected proteins between small and large vesicles. Only 17.4% of these proteins were present in both fractions, whereas 61.5% were unique to smaller vesicles and only 3.7% were exclusively found in the larger ones. Importantly, the pro-resolving protein mediators Annexin A1 and Galectins 1 and 3 were only detected in small vesicles. Lipidomic studies, on the other hand, showed that small vesicles contained higher levels of eicosanoids than large ones and, although all of them incorporated the drugs and molecules investigated, small vesicles were more efficiently loaded with PUFA and the large ones with aspirin, LXA4 and resolvin D1. Importantly, our data indicate that the vesicles contain all necessary enzymatic components for the de novo generation of eicosanoids from fatty acid precursors, including pro-inflammatory agents, suggesting that their cargo should be carefully tailored to avoid interference with their therapeutic purpose. Altogether, these results support the idea that both small and large EVs from auditory HEI-OC1 cells could be used as nanocarriers for anti-inflammatory drugs and pro-resolving mediators. [ABSTRACT FROM AUTHOR]

Objectives: Inflammation is crucial for the pathogenesis of acquired sensorineural hearing loss, but the precise mechanism involved remains elusive. Among a number of inflammatory mediators, tumor necrosis factor-alpha (TNF- α) plays a pivotal role in cisplatin ototoxicity. However, TNF-α alone is cytotoxic to cochlear sensory cells only at the extremely high concentrations, suggesting the involvement of other factors that may sensitize cells to TNF-α cytotoxicity. Since interferon gamma (IFN-γ) importantly contributes to the cochlear inflammatory processes, we aim to determine whether and how IFN-γ affects TNF-α cytotoxicity to cochlear sensory cells. Methods: TNF-α expression was determined with western blotting in RSL cells and immunolabeling of mouse temporal bone sections. HEI-OC1 cell viability was determined with MTT assays, cytotoxicity assays, and cytometric analysis with methylene blue staining. Cochlear sensory cell injury was determined in the organotypic culture of the mouse organ of Corti. Results: Spiral ligament fibrocytes were shown to upregulate TNF-α in response to pro-inflammatory stimulants. We demonstrated IFN-γ increases the susceptibility of HEI-OC1 cells to TNF-α cytotoxicity via JAK1/2-STAT1 signaling. TNFR1-mediated Caspase-1 activation was found to mediate the sensitization effect of IFN-γ on TNF-α cytotoxicity. The combination of IFN-γ and TNF-α appeared to augment cisplatin cytotoxicity to cochlear sensory cells ex vivo. Conclusions: Taken together, these findings suggest the involvement of IFN-γ in the sensitization of cochlear cells to TNF-α cytotoxicity, which would enable us to better understand the complex mechanisms underlying inflammation-mediated cochlear injury. [ABSTRACT FROM AUTHOR]

Cochlear inflammatory response to various environmental insults, including acoustic and ototoxic overexposures, has been increasingly become a topic of interest. As the immune response is associated with both pathology and protection, targeting specific components of the immune response is expected to dissect the relationships between cellular damage and inflammation-associated protection and repair in the cochlea. Duffy antigen receptor for chemokines (DARC) is a member of a group of atypical chemokine receptors, and essential for chemokine-regulated leukocyte/neutrophil trafficking during inflammation. Previous studies have reported that Darc deficiency alters chemokine bioavailability and leukocyte homeostasis, leading to significant anti-inflammatory effects in tissues following injury. In this study, we have used Darc knockout mice to determine the impact of a deficiency in this gene on cochlear development, as well as function in cochlea subjected to various stresses. We observed that DARC is not required for normal development of cochlear function, as evidenced by typical hearing sensitivity in juvenile Darc-KO mice, as compared to wild type (WT) C57BL/6 mice. However, Darc-KO mice exhibited improved hearing recovery after intense noise exposure when compared to wild-type. The auditory brainstem response (ABR) threshold shift between KO and WT mice was most obvious at 1-week post-noise exposure. At cochlear locations above the frequency range of the energy band of damaging noise, both hair cell survival and ribbon synapse density were improved in Darc deficient animals. In addition, the mRNA levels of some major inflammatory effectors, including Mcp-1 and Gdf15, were altered in Darc-KO mice compared to control mice at 1, 3 and 7 days post-noise exposure. These data collectively suggest that the normal Darc-dependent inflammatory response slows down the process of hearing recovery, and exacerbates cellular damage in the cochlea after noise exposure.

Cochlear inflammatory response to various environmental insults, including acoustic and ototoxic overexposures, has been increasingly become a topic of interest. As the immune response is associated with both pathology and protection, targeting specific components of the immune response is expected to dissect the relationships between cellular damage and inflammation-associated protection and repair in the cochlea. Duffy antigen receptor for chemokines (DARC) is a member of a group of atypical chemokine receptors, and essential for chemokine-regulated leukocyte/neutrophil trafficking during inflammation. Previous studies have reported that Darc deficiency alters chemokine bioavailability and leukocyte homeostasis, leading to significant anti-inflammatory effects in tissues following injury. In this study, we have used Darc knockout mice to determine the impact of a deficiency in this gene on cochlear development, as well as function in cochlea subjected to various stresses. We observed that DARC is not required for normal development of cochlear function, as evidenced by typical hearing sensitivity in juvenile Darc-KO mice, as compared to wild type (WT) C57BL/6 mice. However, Darc-KO mice exhibited improved hearing recovery after intense noise exposure when compared to wild-type. The auditory brainstem response (ABR) threshold shift between KO and WT mice was most obvious at 1-week post-noise exposure. At cochlear locations above the frequency range of the energy band of damaging noise, both hair cell survival and ribbon synapse density were improved in Darc deficient animals. In addition, the mRNA levels of some major inflammatory effectors, including Mcp-1 and Gdf15, were altered in Darc-KO mice compared to control mice at 1, 3 and 7 days post-noise exposure. These data collectively suggest that the normal Darc-dependent inflammatory response slows down the process of hearing recovery, and exacerbates cellular damage in the cochlea after noise exposure. [ABSTRACT FROM AUTHOR]

• Cochlear implantation initiates a cochlear innate immune response with accumulation of macrophages • The chronic presence of an indwelling cochlear implant is more important than surgical trauma in contributing to the persistent cochlear immune response • Electric stimulation had no observable effect the cochlear innate immune response Cochlear implantation is an effective auditory rehabilitation strategy for those with profound hearing loss, including those with residual low frequency hearing through use of hybrid cochlear implantation techniques. Post-mortem studies demonstrate the nearly ubiquitous presence of intracochlear fibrosis and neo-ossification following cochlear implantation. Current evidence suggests post-implantation intracochlear fibrosis is associated with delayed loss of residual acoustic hearing in hybrid cochlear implant (CI) recipients and may also negatively influence outcomes in traditional CI recipients. This study examined the contributions of surgical trauma, foreign body response and electric stimulation to intracochlear fibrosis and the innate immune response to cochlear implantation and the hierarchy of these contributions. Normal hearing CX3CR1+/GFP mice underwent either round window opening (sham), acute CI insertion or chronic CI insertion with no, low- or high-level electric stimulation. Electric stimulation levels were based on neural response telemetry (NRT), beginning post-operative day 7 for 5 h per day. Subjects (n=3 per timepoint) were sacrificed at 4 h, 1,4,7,8,11,14 and 21 days. An unoperated group (n=3) served as controls. Cochleae were harvested at each time-point and prepared for immunohistochemistry with confocal imaging. The images were analyzed to obtain CX3CR1+ macrophage cell number and density in the lateral wall (LW), scala tympani (ST) and Rosenthal's canal (RC). A ST peri-implant cellular infiltrate and fibrosis occurred exclusively in the chronically implanted groups starting on day 7 with a concurrent infiltration of CX3CR1+ macrophages not seen in the other groups. CX3CR1+ macrophage infiltration was seen in the LW and RC in all experimental groups within the first week, being most prominent in the 3 chronically implanted groups during the second and third week. The cochlear immune response was most prominent in the presence of chronic cochlear implantation, regardless of electric stimulation level. Further, the development of intracochlear ST fibrosis was dependent on the presence of the indwelling CI foreign body. An innate immune response was evoked by surgical trauma alone (sham and acute CI groups) to a lesser degree. These data suggest that cochlear inflammation and intrascalar fibrosis after cochlear implantation are largely dependent on the presence of a chronic indwelling foreign body and are not critically dependent on electrical stimulation. Also, these data support a role for surgical trauma in inciting the initial innate immune response. [ABSTRACT FROM AUTHOR]

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-1β), 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. [ABSTRACT FROM AUTHOR]

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 (Ktrans). 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. Ktrans 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. J. Magn. Reson. Imaging 2014;39:150-161. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Background: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss., Methods: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context., Findings: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice., Interpretation: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss., Funding: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine., Competing Interests: Declaration of interests The authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

We sought to determine the distribution of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and lymphocytes in the cochlea of mice infected with murine cytomegalovirus (MCMV). For this purpose, 16 newborn mice were divided equally into model and control (uninfected) groups. In model group, 10 μl of MCMV was injected into the brain of each mouse whereas in control group, 10 μl of physiological saline was injected. Fourteen days after the injection of MCMV, the auditory brainstem response audiometry was performed. Later, the mice were killed and the acoustic capsule samples were collected for polymerase chain reaction analysis, histopathological and immunohistochemical studies. Compared with control mice, the incubation period was longer ( F = 13.797; P = 0.003) and the amplitude was lower ( F = 5.095; P = 0.043) in model group mice. It indicated that the intracerebral injection of MCMV caused hearing loss in mice. Histopathological examination of cochleas revealed increased levels of lymphocytic infiltration in the membrana vestibularis. Higher levels of TNF-α and IL-6 in scala tympani were detected by immunohistochemical staining. Taken together, the hearing loss in mice could be related with the inflammatory changes occurring in cochlea after inception of MCMV infection. [ABSTRACT FROM AUTHOR]

Objective: To investigate the role of magnetic resonance imaging (MRI) in the diagnosis of sudden sensorineural hearing loss (SSNHL). Methods: Fifty-four consecutive patients affected by SSNHL were investigated using brain MRI. MRI was performed with an eight-channel phased-array head coil to study the entire audiovestibular pathway and the whole brain. The protocol study consisted of a high-resolution study of the temporal bone, internal auditory canal (IAC}, cerebellopontine angle (CPA), and brainstem combining 2 mm thin-slice axial T2-weighted two-dimensional fast spin echo (FSE) and fluid-attenuated inversion recovery (FLAIR) sequences, pre- and postcontrast (gadolinium-diathyienetriamine pentaacatic acid) administration fat-suppressed axial T1-weighted two-dimensional FSE sequences, and a T2*-weighted three-dimensional Fourier transformation-constructive interference in steady state sequence (FT-CISS), with 0.4 mm ultrathin partitions. The rest of the brain was studied with a 4 mm axial T2-weighted FLAIR sequence. Results: Thirty-one of 54 (57%) cases of SSNHL presented with MRI abnormalities. In 6 of 54 cases, the detected abnormality was directly correlated to the clinical picture (2 labyrinthine hemorrhage, 1 cochlear inflammation, 1 acoustic neuroma, 1 arachnoid cyst of the CPA, and 1 case of white matter lesions in the pons, compatible with demyelinating plaques along the central audiovestibular nervous pathway, as the first expression of multiple sclerosis). Conclusions: An extensive MRI study of the audiovestibular nervous pathway and of the whole brain, pre- and postparamagnetic contrast administration, is recommended to rule out the wide spectrum of abnormalities that can cause SSNHL. [ABSTRACT FROM AUTHOR]

Objectives: The involvement of transforming growth factor β (TGF-β), a strong mediator of fibrogenesis, during cochlear immune responses was investigated. Methods: An inner ear adaptive immune response to antigen was created in mice that were painlessly sacrificed 3 to 48 hours and 7 days after initiation of the immune response. The cochleas were assayed by immunocytochemistry for TGF-β and latency-associated peptide (LAP). Results: We found LAP expressed in normal cochleas and the endolymphatic sac, in the small round cells in the cochlear scalae and the mesothelial cells under the basilar membrane, and in the endolymphatic sac perisaccular area. We found TGF-β expressed in infiltrated, inflammatory cells in the scalae and the endolymphatic sac lumen 3 hours after cochlear antigen challenge. At this time, LAP immunoreactivity was decreased. This rapid shift in immunoreactivity provides evidence for activation of TGF-β during an immune response. This reversal of expression persisted for 48 hours, but conditions reverted to normal after 7 days. Surgical controls did not show TGF-β expression. Conclusions: We conclude that TGF-β activation occurs in the early phase of a cochlear adaptive immune response and is down-regulated as the response resolves. This finding suggests that the process of cochlear fibrosis starts early and that proper treatment could prevent cochlear fibrosis. [ABSTRACT FROM AUTHOR]

The inner ear is capable of rapidly mounting an immune response that can ultimately lead to cochlear degeneration and permanent hearing loss. The role of the endolymphatic sac in this immune process is not clear. In order to investigate the cytokine expression of cells within the endolymphatic sac, a secondary inner ear immune response to keyhole limpet hemocyanin (KLH) was created in mice. The animals were sacrificed 3-48 h and 7 days following initiation of the immune response. The cochleas and endolymphatic sacs were assayed by immunocytochemistry for IL-1beta, TNFalpha, and IL-6. Three hours after KLH challenge of the scala tympani, the perisaccular tissue of the endolymphatic sac contained more inflammatory cells than the scala tympani or endolymphatic sac lumen. Only a few of these cells, however, expressed the proinflammatory cytokines IL-1beta and TNFalpha between 3 and 12 h after KLH injection. On the other hand, TNFalpha, which plays an important role in the cochlear secondary immune response, was expressed in cells in the endolymphatic sac lumen. The maximum percentage of cells expressing TNFalpha was seen later than in the scala tympani. Animals treated with systemic injection of the TNF blocker, etanercept, showed a reduction in the number of cells in the endolymphatic sac lumen. It is concluded that the cells in the endolymphatic sac lumen contribute to the amplification of the adaptive immune response by expressing TNFalpha, while the infiltration of cells into the perisaccular connective tissue is part of the nonspecific, innate, cochlear immune response. [ABSTRACT FROM AUTHOR]