Perivascular-resident macrophage-like melanocytes in the inner ear are essential for the integrity of the intrastrial fluid-blood barrier.

The microenvironment of the cochlea is maintained by the barrier between the systemic circulation and the fluids inside the stria vascularis. However, the mechanisms that control the permeability of the intrastrial fluid-blood barrier remain largely unknown. The barrier comprises endothelial cells connected to each other by tight junctions and an underlying basement membrane. In a recent study, we found that the intrastrial fluid-blood barrier also includes a large number of perivascular cells with both macrophage and melanocyte characteristics. The perivascular-resident macrophage-like melanocytes (PVM/Ms) are in close contact with vessels through cytoplasmic processes. Here we demonstrate that PVM/Ms have an important role in maintaining the integrity of the intrastrial fluid-blood barrier and hearing function. Using a cell culture-based in vitro model and a genetically induced PVM/M-depleted animal model, we show that absence of PVM/Ms increases the permeability of the intrastrial fluid-blood barrier to both low- and high-molecular-weight tracers. The increased permeability is caused by decreased expression of pigment epithelial-derived factor, which regulates expression of several tight junction-associated proteins instrumental to barrier integrity. When tested for endocochlear potential and auditory brainstem response, PVM/M-depleted animals show substantial drop in endocochlear potential with accompanying hearing loss. Our results demonstrate a critical role for PVM/Ms in regulating the permeability of the intrastrial fluid-blood barrier for establishing a normal endocochlear potential hearing threshold.

In silico Single-Cell Analysis of Steroid-Responsive Gene Targets in the Mammalian Cochlea.

BACKGROUND: Treatment of many types of hearing instability in humans, including sudden sensorineural hearing loss, Meniere's disease, and autoimmune inner ear disease, rely heavily on the utilization of corticosteroids delivered both by oral and transtympanic routes. Despite this use, there is heterogeneity in the response to treatment with corticosteroids in humans with these diseases. The mechanisms by which corticosteroids exert their effect and the cell types in which they exert their effects in the inner ear remain poorly characterized. In this study, we localize steroid-responsive genes to cochlear cell types using previously published transcriptome datasets from the mammalian cochlea. METHODS: Steroid-responsive genes were localized to specific cochlear cell types using existing transcriptome datasets from wild-type mammalian cochlea exposed to systemic and transtympanic steroids, as well as previously published single-cell and single-nucleus RNA-sequencing datasets from the mammalian cochlea. Gene ontology (GO) analysis of differentially expressed genes (DEGs) was performed using PANTHER to investigate cellular processes implicated in transtympanic vs. systemic steroid action in the cochlea. RESULTS: Steroid-responsive genes were localized to specific cell types and regions in the cochlea including the stria vascularis, organ of Corti, and spiral ganglion neurons (SGN). Analyses demonstrate differential prevalence of steroid-responsive genes. GO analysis demonstrated steroid-responsive DEGs in the SGN to be associated with angiogenesis, apoptosis, and cytokine-mediated anti-inflammatory pathways. CONCLUSIONS: Single-cell and single-nucleus transcriptome datasets localize steroid-responsive genes to specific regions in the cochlea. Further study of these regionally-specific steroid-responsive genes may provide insight into the mechanisms of and clinical response to corticosteroids in diseases of hearing instability.

Blocking the glucocorticoid receptor with RU-486 does not prevent glucocorticoid control of autoimmune mouse hearing loss.

BACKGROUND/AIMS: Glucocorticoids effectively manage autoimmune hearing loss, although the cochlear mechanisms involved are unknown. Previous studies of steroid-responsive hearing loss in autoimmune (lupus) mice showed glucocorticoids and mineralocorticoids were equally effective, suggesting the ion homeostasis functions of glucocorticoids may be as relevant as immunosuppression for control of autoimmune-induced inner ear disease. Therefore, to better characterize the role of the glucocorticoid receptor in autoimmune hearing loss therapy, its function was blocked with the antagonist RU-486 (mifepristone) during glucocorticoid (prednisolone) treatments. METHODS: Following baseline auditory brainstem response (ABR) thresholds, MRL/MpJ-Fas(lpr) autoimmune mice were implanted with pellets providing combinations of 1.25 mg/kg of RU-486, 4 mg/kg of prednisolone, or their respective placebos. After 1 month, animals were retested with ABR and blood was collected for immune complex analyses. RESULTS: Mice receiving no prednisolone (placebo + placebo and placebo + RU-486) showed continued declines in hearing. On the other hand, mice receiving prednisolone (prednisolone + placebo and prednisolone + RU-486) had significantly better hearing (p < 0.05) than the non-prednisolone groups. Immune complexes were significantly elevated in the placebo + RU-486 group, suggesting RU-486 effectively blocked glucocorticoid receptor-mediated immune suppression. These results showed that blockage of the glucocorticoid receptor with RU-486 did not prevent prednisolone's effects in the ear, suggesting its ion homeostasis actions via the mineralocorticoid receptor were more relevant in hearing control. CONCLUSION: The mineralocorticoid receptor-mediated actions of glucocorticoids are potentially relevant in steroid-responsive hearing disorders, implying disrupted cochlear ion transport functions may underlie the vascular problems proposed in some forms of immune-mediated hearing loss.

Advances in Auditory and Vestibular Medicine.

Auditory and Vestibular medicine is becoming more accepted as a specialty of its own, Medical NeurOtology. Recent advances in the field have been instrumental in the understanding of the scientific foundations, pathophysiology, clinical approach and management of patients with hearing and vestibular disorders. This paper will review these advances.

Intratympanically Delivered Steroids Impact Thousands More Inner Ear Genes Than Systemic Delivery.

OBJECTIVES: Glucocorticoids are given for sensorineural hearing loss, but little is known of their molecular impact on the inner ear. Furthermore, in spite of claims of improved hearing recovery with intratympanic delivery of steroids, no studies have actually documented the inner ear molecular functions that are enhanced with this delivery method. METHODS: To assess steroid-driven processes in the inner ear, gene chip analyses were conducted on mice treated systemically with the glucocorticoids prednisolone or dexamethasone or the mineralocorticoid aldosterone. Other mice were given the same steroids intratympanically. Inner ears were harvested at 6 hours and processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34 000 genes. Results were statistically analyzed for up or down expression of each gene against control (untreated) mice. RESULTS: Analyses showed approximately 17 500 genes are normally expressed in the inner ear and steroids alter expression of 55% to 82% of these. Dexamethasone changed expression of 9424 (53.9%) inner ear genes following systemic injection but 14 899 ear genes (85%) if given intratympanically. A similar pattern was seen with prednisolone, as 7560 genes were impacted by oral delivery and 11 164 genes (63.8%) when given intratympanically. The mineralocorticoid aldosterone changed expression of only 268 inner ear genes if given orally, but this increased to 10 124 genes (57.9%) if injected intratympanically. Furthermore, the glucocorticoids given actually impacted more inner ear genes via the mineralocorticoid receptor than the glucocorticoid receptor. CONCLUSIONS: Thousands of inner ear genes were affected by steroids, and this number increased significantly if steroids were delivered intratympanically. Also, the impact of glucocorticoids on inner ear mineralocorticoid functions is more substantial than previously known. Thus, the application of therapeutic steroids for hearing loss needs to be reassessed in light of their more comprehensive impact on inner ear genes. Furthermore, simply ascribing the efficacy of steroids to immunosuppression no longer appears to be warranted.

Control of chronic otitis media and sensorineural hearing loss in C3H/HeJ mice: glucocorticoids vs mineralocorticoids.

OBJECTIVE: The impact of glucocorticoids and mineralocorticoids on chronic otitis media (COM) in toll-like receptor 4-deficient C3H/HeJ mice was investigated. STUDY DESIGN: To evaluate control of COM by steroids with differences in their anti-inflammatory (prednisolone, dexamethasone), and fluid absorption functions (fludrocortisone, aldosterone). A minimum sample size of five animals for each group was required based on power analysis calculations. Sample sizes ranged from 7 to 17 mice per treatment group. SUBJECTS AND METHODS: Auditory brain stem response (ABR) thresholds were performed at baseline, 2 weeks and 4 weeks. Histopathologic test results were evaluated on all mice ears at the end of the study. RESULTS: Analysis of variance (ANOVA) of ABR threshold change showed significant treatment effects (P < 0.05) by both steroid types at all time intervals and ABR frequencies except 4 weeks/8 kHz. Histologic assessment showed prednisolone-treated mice (62%) had a higher rate of clearance of middle and inner ear inflammation than control mice (4%). CONCLUSION: It was concluded that steroid treatments can improve the physiology of chronic middle and inner ear disease seen with COM.

Gram-negative pathogen Klebsiella oxytoca is associated with spontaneous chronic otitis media in Toll-like receptor 4-deficient C3H/HeJ mice.

CONCLUSION: This report confirms the presence of gram-negative Klebsiella bacteria in the middle ear of the C3H/HeJ mouse by culture, polymerase chain reaction (PCR), and electron microscopy. Identification of the bacterial pathogen supports the C3H/HeJ mouse as an excellent model for spontaneous chronic otitis media and its effects on the middle and inner ear. OBJECTIVES: The C3H/HeJ mouse has a single amino acid substitution in its Toll-like receptor 4, making it insensitive to endotoxin and suppressing initiation of the innate immune system. This study explored the bacteriology of the resultant middle ear infection by culture, PCR, histology, and electron microscopy. MATERIALS AND METHODS: Twelve-month-old C3H/ HeJ mice were screened positive for spontaneous otitis media. Tympanocentesis and blood cultures of mice were carried out under sedation. Middle ear aspirate material and blood samples were then sent for culture and PCR. Mice were then sacrificed for bright-field and electron microscopy analysis. RESULTS: All tympanocentesis and blood specimens grew gram-negative Klebsiella oxytoca, which was confirmed by PCR. Histopathology confirmed an intense inflammatory reaction and gram-negative bacteria in the middle and inner ears. Electron microscopy of the middle ears revealed abundant rod-shaped Klebsiella bacteria, both free and being engulfed by neutrophils.

Glucocorticoid impact on cochlear function and systemic side effects in autoimmune C3.MRL-Faslpr and normal C3H/HeJ mice.

Glucocorticoids are effective in reversing hearing loss, but their severe side effects limit long term management of many ear disorders. A clearer understanding of these side effects is critical for prolonged therapeutic control of hearing and vestibular dysfunction. Therefore, this study characterized the impact of the glucocorticoid prednisolone on cochlear dysfunction and systemic organ systems in C3.MRL-Fas(lpr) autoimmune mice and their normal C3H/HeJ parent strain. Following 3 months of treatment, autoimmune mice had better auditory thresholds and improved hematocrits, anti-nuclear antibodies, and immune complexes. Steroid treatment also lowered body and spleen weights, both of which rise with systemic autoimmune disease. Steroid treatment of the normal C3H/HeJ mice significantly elevated their blood hematocrits and lowered their body and spleen weights to abnormal levels. Thus, systemic autoimmune disease and its related hearing loss in C3.MRL-Fas(lpr) mice are steroid-responsive, but normal hemopoiesis and organ functions can be significantly compromised. This mouse model may be useful for studies of the detrimental side effects of steroid treatments for hearing loss.

Otoprotective effects of dexamethasone in the management of pneumococcal meningitis: an animal study.

OBJECTIVE: To determine whether treating pneumococcal meningitis with a combined antibiotic and steroid regime will prevent cochlear damage, a common pneumococcal meningitis side effect. STUDY DESIGN: Prospective animal study. METHODS: Gerbils were randomly assigned to three experimental groups. Animals in group 1 received intrathecal saline injections. Animals in groups 2 and 3 received intrathecal injections of Streptococcus pneumoniae to induce meningitis. Group 2 was treated for 7 days with intraperitoneal penicillin injections (48,000 units). Animals from group 3 received intraperitoneal dexamethasone (0.5 mg/kg) injections for 4 days in addition to 7 days of intraperitoneal penicillin. Three months after the meningitis was induced, the animals' cochlear functions were determined using auditory brainstem responses (ABRs). After measuring cochlear function, the animals were sacrificed for cochlear histopathology. Spiral ganglion cell densities at Rosenthal's canal were determined. RESULTS: ABR thresholds were significantly elevated in animals from group 2 when compared with the animals in groups 1 and 3 (P < .05). ABR thresholds for animals from group 3 and group 1 were similar (P > .05). Damage of cochlear structures was detected in animals from group 2. The degree of the damage varied: one animal in group 2 had no identifiable hair cells and pillar cells and showed damage of the tectorial membrane. Spiral ganglion density in the basal turn was significantly less in animals from group 2 when compared with controls (P < .05). Although spiral ganglion cell density was less in the dexamethasone-treated group (group 3) when compared with group 1 (control group), but greater than observed in animals treated with antibiotics only (group 2), the differences were statistically not significant (P > .5). Nuclear diameters of the spiral ganglion cells decreased on average from 7.24 +/- 0.48 microm (group 1) to 6.28 +/- 0.76 microm (group 3, animals that received dexamethasone) to 5.57 +/- 0.82 microm (group 2, animals that received antibiotics only). Differences were significant (P < .05). Differences in stria vascularis thickness were not significant among the animals. CONCLUSION: Dexamethasone has a protective effect on the cochlea when given together with antibiotics in the treatment of pneumococcal meningitis.

Cochlear cytokine gene expression in murine acute otitis media.

OBJECTIVE: Recurrent acute otitis media (AOM) causes sensorineural hearing loss by unknown mechanisms. It is widely accepted that inflammatory cytokines diffuse across the round window membrane to exert cytotoxic effects. This study addresses whether inner ear cells are capable of expressing genes for inflammatory cytokines. STUDY DESIGN: The authors conducted a prospective animal study. METHODS: BALB/C mice underwent transtympanic injection of heat-killed Haemophilus influenzae to create an acute inflammatory response. These mice were compared with a control group in addition to a group of uninjected mice found to have otomicroscopic changes consistent with persistent or chronic otitis media. The cochleas of these mice were obtained, their RNA harvested, and cytokine gene expression analyzed using prefabricated cDNA arrays. RESULTS: Four groups of mice (control, 3-day postinjection, 7-day postinjection, and mice with chronic otitis media) with five mice in each group were analyzed. Numerous classes of genes were found to be upregulated or downregulated by more than twofold. Some genes differed from control mice by more than 10-fold. These genes included numerous fibroblast growth factors, interleukins, tumor necrosis factors, and colony-stimulating factors. CONCLUSION: The genes of numerous inflammatory cytokines are either up- or downregulated by murine inner ear cells in response to either acute or chronic inflammation of the middle ear. This study provides a novel site of production of cytokines that may be responsible for the damage seen in sensorineural hearing loss.

Cochlear cytokine gene expression in murine chronic otitis media.

OBJECTIVE: To investigate chronic otitis media (COM) induction of cochlear cytokine genes. STUDY DESIGN: RNA from cochleas of five C3H/HeJ mice with and without COM was isolated for cytokine expression in gene arrays. Immunohistochemistry was performed for the protein products of up-regulated genes to confirm their expression in cochlear tissues. RESULTS: Cochleas from COM mice showed increased expression of 29 genes (>2x normal) and decreased expression of 19 genes (<0.5x normal). Cytokines expressed were largely those related to inflammation and tissue remodeling. Cochlear immunohistochemistry confirmed the presence of numerous cytokines, as well as NF-kB, a major inflammatory transcription factor that drives cytokine expression. CONCLUSION: COM causes elevated levels of cochlear cytokine mRNA, which demonstrates that inner ear tissues are capable of NF-kB activation and cytokine production. This may be another mechanism of otitis media-induced cochlear cytotoxicity in addition to that caused by migration of inflammatory cytokines from the middle ear. SIGNIFICANCE: Cochlear tissues are capable of mounting an immunological response to middle ear inflammatory stimuli.

Mineralocorticoid receptor mediates glucocorticoid treatment effects in the autoimmune mouse ear.

The standard treatment for many hearing disorders is glucocorticoid therapy, although the cochlear mechanisms involved in steroid-responsive hearing loss are poorly understood. Cochlear dysfunction in autoimmune mice has recently been shown to be controlled with the mineralocorticoid aldosterone as effectively as with the glucocorticoid prednisolone. Because aldosterone regulates sodium, potassium, and other electrolyte homeostasis, this implied the restoration of hearing with the mineralocorticoid was due to its impact on cochlear ion transport, particularly in the stria vascularis. This also suggested glucocorticoids may be controlling hearing recovery in part through their binding to the mineralocorticoid receptor in addition to their glucocorticoid receptor-mediated anti-inflammatory and immunosuppressive functions. Therefore, the aim of the present study was to better delineate the role of the mineralocorticoid receptor in steroid control of hearing in the autoimmune mouse. Spironolactone, a mineralocorticoid receptor antagonist, was administered to MRL/MpJ-Fas(lpr) autoimmune mice in combination with either aldosterone or prednisolone to compare their hearing and systemic disease with mice that received either steroid alone. ABR thresholds showed either aldosterone or prednisolone alone preserved hearing in the mice, but spironolactone prevented both steroids from maintaining normal cochlear function. This suggested both steroids are preserving hearing through the mineralocorticoid receptor within the ear to regulate endolymph homeostasis. The spironolactone treatment did not block normal glucocorticoid receptor-mediated immune-suppression functions because mice receiving prednisolone, either with or without spironolactone, maintained normal body weights, hematocrits, and serum immune complexes. Thus, reducing systemic autoimmune disease was not sufficient to control hearing if mineralocorticoid receptor-mediated functions were blocked. It was concluded the inner ear mineralocorticoid receptor is a significant target of glucocorticoids and a factor that should be considered in therapeutic treatments for steroid-responsive hearing loss.

Evaluation of the mouse model for acute otitis media.

Various animal models have been employed for otitis media research. The mouse has been studied less, in spite of its many advantages. To better understand the suitability of the mouse for studies of otitis media, an evaluation was made of its middle ear inflammatory processes following inoculation with heat-killed Streptococcus pneumoniae (strain 6A), one of the three most common bacteria to cause otitis media in the human. A total of 94 BALB/c mice were injected transtympanically with three concentrations of heat-killed bacteria (10(4), 10(6), and 10(9) organisms per ml) and inflammation evaluated with both histologic examination and auditory brainstem response audiometry. Dose-related measures of the time course of inflammation showed it was maximal at 3 days. PBS-injected control mice also demonstrated some degree of middle ear inflammation. Therefore, inflammation measures from PBS injected mice were used as the threshold above which histologic inflammatory changes would be considered a response to bacteria. These quantitative comparisons of bacterial and PBS inoculations revealed the most significant middle ear measures of inflammation were amount of fluid in the middle ear, tympanic membrane thickness, and number of inflammatory cells. The induction of middle ear inflammation in the mouse demonstrated the applicability of this model for investigations of otitis media.

C3H/HeJ mouse model for spontaneous chronic otitis media.

OBJECTIVES/HYPOTHESIS: Chronic otitis media is a significant clinical problem. Understanding the mechanisms of chronic otitis media is critical for its control. However, little is known of these processes as a result of lack of animal models of spontaneous otitis media. The C3H/HeJ mouse has a single amino acid substitution in its toll-like receptor 4 (TLR4), making it insensitive to endotoxin. As a result, these mice cannot clear Gram-negative bacteria. The chronically inflamed middle ear in this animal provides us the opportunity to study spontaneous chronic otitis media. STUDY DESIGN AND METHODS: Otoscopy and auditory brain response (ABR) evaluation of C3H/HeJ mice at 3, 5, 7, 9, and 11 months were carried out under sedation. At 12 months of age, mice were killed and histologic analysis of the middle ear, inner ear, and eustachian tube was carried out. RESULTS: Tympanic membrane visualization and ABR thresholds in 7- to 8-month-old C3H/HeJ mice showed that approximately half developed middle and inner ear disease spontaneously. The significant elevation of thresholds suggested a sensorineural component in addition to the conductive loss. Middle and inner ear histology showed some degree of middle and inner ear inflammation in half the mice, paralleling the ABR data. CONCLUSIONS: The histopathologic changes reported here in the C3H/HeJ mouse model of chronic otitis media have been reported in human chronic otitis media. This spontaneous model of chronic otitis media will be valuable for the characterization of middle and inner ear inflammatory disease processes that are induced by middle ear infections.

Mouse models of otitis media.

PURPOSE OF REVIEW: Otitis media is one of the most prevalent inflammatory diseases in the pediatric population. The personal and societal costs for otitis media are significant. Problems arising from antibiotic use have led to considerable animal research efforts to better understand the mechanisms of acute otitis media and to develop new strategies for its prevention and treatment. RECENT FINDINGS: Various animal models induce acute otitis media from a variety of interventions, including direct injection of whole bacteria or their products into the middle ear. The mouse model has begun to emerge as a model for otitis media. The mouse affords many advantages for in-vivo research, including ease of genetic manipulation, availability of numerous inbred and transgenic strains, and an extensively studied immune system. Experimental reagents for cellular and molecular studies are widely available for the mouse. The mouse is an excellent model for investigating the genetics and molecular bases for otitis media due to the extensive understanding of the mouse genome. SUMMARY: With the increased availability of knockout and transgenic mice, and the large amount of data to indicate that human disease is accurately modeled in the mouse, the mouse model is increasingly becoming a model of choice.

Intratympanic injection of dexamethasone: time course of inner ear distribution and conversion to its active form.

HYPOTHESIS: Intratympanically injected dexamethasone 21-phosphate is converted to its active form dexamethasone in the inner ear and follows the distribution of the glucocorticoid receptor. BACKGROUND: Although dexamethasone is routinely delivered intratympanically for hearing loss, we know little of its inner ear pharmacokinetics. Dexamethasone 21-phosphate is the pharmaceutical compound available for injection, but it must be converted to its biologically active form (dexamethasone) to bind to the glucocorticoid receptor. Therefore, the current study was conducted to determine the time course of dexamethasone 21-phosphate movement from the middle ear into the inner ear, its conversion to dexamethasone, and the distribution of both forms relative to the glucocorticoid receptor. METHODS: BALB/c mice were injected intratympanically with the prodrug dexamethasone 21-phosphate and inner ears collected at postinjection times ranging from 5 minutes to 7 days. Ears were immunohistochemically stained for dexamethasone 21-phosphate, dexamethasone, and the glucocorticoid receptor. RESULTS: Both forms of dexamethasone were seen in the inner ear within 15 minutes, reaching their highest staining intensity at 1 hour. Neither drug was seen after 24 hours. The strongest staining occurred in the spiral ligament, organ of Corti, spiral ganglion, and vestibular sensory epithelia. Distribution of the drug paralleled locations of the glucocorticoid receptor except in the stria vascularis marginal cells, which stained heavily for the receptor but not the drug. CONCLUSION: Dexamethasone rapidly travels from the middle ear into the inner ear and converts to its active form. The drug distribution follows that of the glucocorticoid receptor. However, it probably has little impact on ear tissues after 24 hours.

Tracheal reconstruction with porcine small intestine submucosa in a rabbit model.

OBJECTIVE: To evaluate the ability of porcine small intestine submucosa (SIS) to 1) maintain airway patency, 2) integrate, 3) prevent granulation tissue formation, and 4) permit mucosalization when used for tracheal reconstruction. Further studies were performed to evaluate the ability of SIS to support neochondrogenesis and investigate the impact of neochondrogenesis on airway patency. STUDY DESIGN: Prospective, controlled animal trial with SIS used with and without a perichondrial flap to reconstruct a tracheal defect in a rabbit model. Functional, histologic and endoscopic analyses were performed. RESULTS: All animals except 1 control animal were without stridor. The SIS graft supported neochondrogenesis, was completely mucosalized and was well integrated into the neotrachea. There was minimal granulation tissue formation. Endoscopic analyses did not reveal a consistent, significant difference in airway patency when SIS, with or without a perichondrial flap, was used for reconstruction. CONCLUSION: SIS can be used to reconstruct a sublethal rabbit tracheal defect with no mortality and minimal morbidity.

Intratympanic Steroid Treatments May Improve Hearing via Ion Homeostasis Alterations and Not Immune Suppression.

HYPOTHESIS: The inner ear (IE) endothelium is capable of responding to therapeutic steroids by altering the local expression of cytokine and ion homeostasis genes that impact inflammation and fluid regulation. BACKGROUND: Glucocorticoids are often given transtympanically for hearing disorders because of their anti-inflammatory effects, but their direct impact on IE ion homeostasis and cytokine gene expression has not been studied. METHODS: The middle ears of Balb/c mice were transtympanically injected with 5 µL of phosphate-buffered saline, prednisolone (Pred), or dexamethasone (Dex). Untreated mice were used as controls. Mice were euthanized at 6, 24, and 72 hours; the cochleas were harvested; and total RNA was isolated from the IE tissues. Expression of eight cytokine genes and 24 ion homeostasis genes was analyzed with quantitative real time reverse transcription polymerase chain reaction. RESULTS: Phosphate-buffered saline caused upregulation of inflammatory cytokine genes that peaked at 6 hours. Surprisingly, Pred and Dex also caused upregulation of most cytokine genes. Interestingly, ion homeostasis genes were predominantly upregulated with Dex and Pred, with Pred having a larger effect. CONCLUSION: In the murine model, intratympanic steroids caused an initial upregulation of inflammatory cytokine genes in the IE, as well as predominant upregulation of ion homeostasis genes. These findings suggest that glucocorticoids do not suppress IE inflammation but rather cause an initial inflammatory response in the IE. Thus, inflammatory gene suppression is not a likely mechanism for their hearing restorative effects. On the other hand, these steroids have a significant mineralocorticoid function, as demonstrated by increased function of ion homeostasis genes, implicating their ionic and fluid regulatory properties as a mechanism for their therapeutic effects.

Control of middle ear inflammatory and ion homeostasis genes by transtympanic glucocorticoid and mineralocorticoid treatments.

HYPOTHESIS: Transtympanic steroid treatment will induce changes in ion homeostasis and inflammatory gene expression to decrease middle ear inflammation due to bacterial inoculation. BACKGROUND: Otitis media is common, but treatment options are limited to systemic antibiotic therapy or surgical intervention. Systemic glucocorticoid treatment of mice decreases inflammation and improves fluid clearance. However, transtympanic delivery of glucocorticoids or mineralocorticoid has not been explored to determine if direct steroid application is beneficial. METHODS: Balb/c mice received transtympanic inoculation of heat-killed Haemophilus influenzae (H flu), followed by transtympanic treatment with either prednisolone or aldosterone. Mice given PBS instead of steroid and untreated mice were used as controls. Four hours after steroid treatment, middle ears were harvested for mRNA extraction and 24 hours after inoculation middle ears were harvested and examined for measures of inflammation. RESULTS: H flu inoculation caused the increased expression of nearly all inflammatory cytokine genes and induced changes in expression of several genes related to cellular junctions and transport channels. Both steroids generally reversed the expression of inflammatory genes and caused ion and water regulatory genes to return to normal or near normal levels. Histologic evaluation of middle ears showed improved fluid and inflammatory cell clearance. CONCLUSION: Improvement in middle ear inflammation was noted with both the glucocorticoid prednisolone and the mineralocorticoid aldosterone. This was due to reversal of inflammation-induced changes in middle ear cytokine genes, as well as those involved in ion and water homeostasis. Because glucocorticoids bind to the mineralocorticoid receptor, but not the reverse, it is concluded that much of the reduction of fluid and other inflammation measures was due to these steroids impact on ion and water transport channels. Further research is necessary to determine if this alternative mineralocorticoid treatment for otitis media will be clinically effective with fewer side effects than glucocorticoids.

Correlative mRNA and protein expression of middle and inner ear inflammatory cytokines during mouse acute otitis media.

Although the inner ear has long been reported to be susceptible to middle ear disease, little is known of the inflammatory mechanisms that might cause permanent sensorineural hearing loss. Recent studies have shown inner ear tissues are capable of expressing inflammatory cytokines during otitis media. However, little quantitative information is available concerning cytokine gene expression in the inner ear and the protein products that result. Therefore, this study was conducted of mouse middle and inner ear during acute otitis media to measure the relationship between inflammatory cytokine genes and their protein products with quantitative RT-PCR and ELISA, respectively. Balb/c mice were inoculated transtympanically with heat-killed Haemophilus influenzae and middle and inner ear tissues collected for either quantitative RT-PCR microarrays or ELISA multiplex arrays. mRNA for several cytokine genes was significantly increased in both the middle and inner ear at 6 h. In the inner ear, these included MIP-2 (448 fold), IL-6 (126 fold), IL-1ß (7.8 fold), IL-10 (10.7 fold), TNFα (1.8 fold), and IL-1α (1.5 fold). The 24 h samples showed a similar pattern of gene expression, although generally at lower levels. In parallel, the ELISA showed the related cytokines were present in the inner ear at concentrations higher by 2-122 fold higher at 18 h, declining slightly from there at 24 h. Immunohistochemistry with antibodies to a number of these cytokines demonstrated they occurred in greater amounts in the inner ear tissues. These findings demonstrate considerable inflammatory gene expression and gene products in the inner ear following acute otitis media. These higher cytokine levels suggest one potential mechanism for the permanent hearing loss seen in some cases of acute and chronic otitis media.