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.

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.

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.

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.

Differential expression of innate immunity genes in chronic rhinosinusitis.

BACKGROUND: Prior research has identified several components of the innate immune system that may play a significant role in chronic rhinosinusitis (CRS), but the role of innate immunity in patients with CRS is poorly understood. The objective of this study was to determine differential expression of innate immunity genes in the mucosa of patients with CRS with nasal polyposis (CRSwNP) and CRS without nasal polyposis (CRSsNP) when compared with controls. METHODS: Control patients (n = 9) and patients with CRS (n = 36) who failed medical management were prospectively enrolled. Ethmoid mucosa samples were harvested during surgery and quantitative real-time polymerase chain reaction was used to determine levels of mRNA expression of Toll-like receptor (TLR) 2 and TLR9 and interleukin-22 receptor (IL-22R). The average change in crossover threshold and fold change were calculated and differences between controls, CRSwNP, and CRSsNP were compared. Statistical analysis was performed using the Kruskall-Wallis and adjusted Mann-Whitney U tests. RESULTS: Patients with CRSwNP (n = 16) and CRSsNP (n = 20) showed lower mean expression of TLR2 (p < 0.05) compared with controls. Patients with CRSsNP showed significantly higher mean expression of IL-22R (p < 0.05) than controls. CONCLUSION: The sinonasal innate immune system may have a significant role in the development of CRS. We found differential expression of innate immune mediators between patients with and without nasal polyposis. These results provide further evidence of disruption of innate immunity at the mucosal level in CRS and highlight differences between polyp- and non-polyp-forming CRS phenotypes at the molecular level. In addition to our knowledge, this is the first report of altered IL-22R expression in CRSsNP patients. This study was a part of the clinical trial NCT01332136 registered in www.clinicaltrials.gov.

Otitis media impacts hundreds of mouse middle and inner ear genes.

OBJECTIVE: Otitis media is known to alter expression of cytokine and other genes in the mouse middle ear and inner ear. However, whole mouse genome studies of gene expression in otitis media have not previously been undertaken. Ninety-nine percent of mouse genes are shared in the human, so these studies are relevant to the human condition. METHODS: To assess inflammation-driven processes in the mouse ear, gene chip analyses were conducted on mice treated with trans-tympanic heat-killed Hemophilus influenza using untreated mice as controls. Middle and inner ear tissues were separately harvested at 6 hours, RNA extracted, and samples for each treatment processed on the Affymetrix 430 2.0 Gene Chip for expression of its 34,000 genes. RESULTS: Statistical analysis of gene expression compared to control mice showed significant alteration of gene expression in 2,355 genes, 11% of the genes tested and 8% of the mouse genome. Significant middle and inner ear upregulation (fold change >1.5, p<0.05) was seen in 1,081 and 599 genes respectively. Significant middle and inner ear downregulation (fold change <0.67, p<0.05) was seen in 978 and 287 genes respectively. While otitis media is widely believed to be an exclusively middle ear process with little impact on the inner ear, the inner ear changes noted in this study were numerous and discrete from the middle ear responses. This suggests that the inner ear does indeed respond to otitis media and that its response is a distinctive process. Numerous new genes, previously not studied, are found to be affected by inflammation in the ear. CONCLUSION: Whole genome analysis via gene chip allows simultaneous examination of expression of hundreds of gene families influenced by inflammation in the middle ear. Discovery of new gene families affected by inflammation may lead to new approaches to the study and treatment of otitis media.

Age-related cochlear cytokine gene expression in the BALB/cJ mouse with systemic versus intratympanic dosing of steroid drugs.

CONCLUSION: Age-related differences in the expression of inflammatory cytokines in the inner ear may contribute to the development of age-related hearing loss (ARHL). OBJECTIVES: ARHL is characterized by tissue remodeling, ischemia, ion homeostasis, and inflammation. Steroid therapy is an otoprotective strategy that likely acts by reducing inflammation. We examined age-related changes in cytokine gene expression in the cochlea of the BALB/cJ mouse model of premature ARHL after systemic or intratympanic steroid delivery. METHODS: 'Young' (2.5-3 months) and 'Old' (5-9 months) mice were treated with dexamethasone or fludrocortisone administered either orally or intratympanically. Cytokine gene expression in cochlear RNA was analyzed using prefabricated cDNA arrays. Old groups were compared to Young groups to identify age-related changes. RESULTS: Down-regulation of a cytokine associated with bone remodeling (SPP1) was observed in the untreated Old group. Numerous genes were up- or down-regulated by more than twofold by steroid treatment, including proinflammatory interleukins (IL-16) and anti-inflammatory cytokines.

Steroid-independent upregulation of matrix metalloproteinase 9 in chronic rhinosinusitis patients with radiographic evidence of osteitis.

BACKGROUND: Chronic sinonasal inflammation is associated with tissue remodeling, such as osteitis, which may be a marker of refractory disease; however, the pathophysiology of osteitis in chronic rhinosinusitis (CRS) is insufficiently understood. METHODS: Ethmoid mucosa and bone samples were obtained from 35 medically refractory CRS patients and 9 control subjects. Quantitative real-time polymerase chain reaction (RT-PCR) was performed separately on bone and mucosa for matrix metalloproteinase 2 and 9 (MMP2, MMP9) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1). Osteitis was classified as mild, moderate, or severe by measuring bone thickness of the maxillary, sphenoid, and ethmoid sinuses on multiplanar computed tomography (CT). Patients were classified based on severity of osteitis and compared to controls. RESULTS: Nine patients demonstrated radiographic evidence of osteitis (mild = 3, moderate/severe = 6). Bone PCR revealed biologically significant upregulation of MMP9 in all patients with CRS, but the magnitude of the upregulation decreased with severity of osteitis. Mucosa PCR showed upregulation of MMP9 in moderate/severe osteitis only. No significant changes were seen in MMP2 or TIMP1 regulation. CONCLUSION: This is the first study to evaluate the role of MMP in the bone and mucosa of patients with sinonasal osteitis. The pattern of expression suggests there may be a time- and tissue-dependent role for MMP9 in the pathophysiology of osteitis. In addition, MMP9 overexpression is seen despite preoperative oral and intranasal steroid use, suggesting that if MMP9 is an important factor in the development of osteitis then steroids may not be the best treatment in prevention of osteitis.

Inner ear tissue remodeling and ion homeostasis gene alteration in murine chronic otitis media.

HYPOTHESIS: Studies were designed to ascertain the impact of chronic middle ear infection on the numerous ion and water channels, transporters, and tissue remodeling genes in the inner and middle ear. BACKGROUND: Permanent sensorineural hearing loss is a significant problem resulting from chronic middle ear disease, although the inner ear processes involved are poorly defined. Maintaining a balanced ionic composition of endolymph in the inner ear is crucial for hearing; thus, it was hypothesized that this may be at risk with inflammation. METHODS: Inner and middle ear RNA collected separately from 6-month-old C3H/HeJ mice with prolonged middle ear disease were subjected to qRT-PCR for 8 common inflammatory cytokine genes, 24 genes for channels controlling ion (sodium, potassium, and chloride) and water (aquaporin) transport, tight junction claudins, and gap junction connexins, and 32 tissue remodeling genes. Uninfected Balb/c mice were used as controls. RESULTS: Significant increase in inner ear inflammatory and ion homeostasis (claudin, aquaporin, and gap junction) gene expression, and both upregulation and downregulation of tissue remodeling gene expression occurred. Alteration in middle ear ion homeostasis and tissue remodeling gene expression was noted in the setting of uniform upregulation of cytokine genes. CONCLUSION: Chronic inflammatory middle ear disease can impact inner ear ion and water transport functions and induce tissue remodeling. Recognizing these inner ear mechanisms at risk may identify potential therapeutic targets to maintain hearing during prolonged otitis media.

Corticosteroid therapy for hearing and balance disorders.

This review addresses the current status of steroid therapies for hearing and vestibular disorders and how certain misconceptions may be undermining the efficacy in restoring normal ear function, both experimentally and clinically. Specific misconceptions addressed are that steroid therapy is not effective, steroid-responsive hearing loss proves an underlying inflammatory problem in the ear, and steroids only have application to the hearing disorders listed below. Glucocorticoid therapy for hearing and balance disorders has been employed for over 60 years. It is recommended in cases of sudden hearing loss, Meniére's disease, immune-mediated hearing loss, and any vestibular dysfunction suspected of having an inflammatory etiology. The predominant steroids employed today are dexamethasone, prednisone, prednisolone, and methylprednisolone. Despite years of use, little is known of the steroid responsive mechanisms in the ear that are influenced by glucocorticoid therapy. Furthermore, meta-analyses and clinical study reviews occasionally question whether steroids offer any benefit at all. Foremost in the minds of clinicians is the immune suppression and anti-inflammatory functions of steroids because of their efficacy for autoimmune hearing loss. However, glucocorticoids have a strong binding affinity for the mineralocorticoid (aldosterone) and glucocorticoid receptors, both of which are prominent in the ear. Because the auditory and vestibular end organs require tightly regulated endolymph and perilymph fluids, this ion homeostasis role of the mineralocorticoid receptor cannot be overlooked in both normal and pathologic functions of the ear. The function of the glucocorticoid receptor is to provide anti-inflammatory and antiapoptotic signals by mediating survival factors.

Mouse middle ear ion homeostasis channels and intercellular junctions.

HYPOTHESIS: The middle ear contains homeostatic mechanisms that control the movement of ions and fluids similar to those present in the inner ear, and are altered during inflammation. BACKGROUND: The normal middle ear cavity is fluid-free and air-filled to allow for effective sound transmission. Within the inner ear, the regulation of fluid and ion movement is essential for normal auditory and vestibular function. The same ion and fluid channels active in the inner ear may have similar roles with fluid regulation in the middle ear. METHODS: Middle and inner ears from BALB/c mice were processed for immunohistochemistry of 10 specific ion homeostasis factors to determine if similar transport and barrier mechanisms are present in the tympanic cavity. Examination also was made of BALB/c mice middle ears after transtympanic injection with heat-killed Haemophilus influenza to determine if these channels are impacted by inflammation. RESULTS: The most prominent ion channels in the middle ear included aquaporins 1, 4 and 5, claudin 3, ENaC and Na(+),K(+)-ATPase. Moderate staining was found for GJB2, KCNJ10 and KCNQ1. The inflamed middle ear epithelium showed increased staining due to expected cellular hypertrophy. Localization of ion channels was preserved within the inflamed middle ear epithelium. CONCLUSIONS: The middle ear epithelium is a dynamic environment with intrinsic mechanisms for the control of ion and water transport to keep the middle ear clear of fluids. Compromise of these processes during middle ear disease may underlie the accumulation of effusions and suggests they may be a therapeutic target for effusion control.

Innate immunity gene single nucleotide polymorphisms and otitis media.

OBJECTIVE: Toll-like receptors (TLR) activate the innate immune system. Single nucleotide polymorphisms (SNPs) in TLR genes are linked to increased susceptibility to infections. TLR4-deficient mice have increased incidence and duration of otitis media. We hypothesize that SNPs in TLR genes are more common in otitis-prone children than in children without a history of otitis media. METHODS: Cases (n=70) included children undergoing surgery for otitis media. Control subjects (n=70) included children undergoing surgery for non-otologic indication. Genomic DNA was extracted from blood samples. RT-PCR genotyping was performed for TLR2 (rs5743708), TLR4 (rs4986790 and rs4986791), TLR9 (rs5743836 & rs187084), and CD14 (rs2569190). RESULTS: There were no significant differences between the groups in family history, day care, smoke exposure, allergies or prevalence of the SNPs. The most common pre-op diagnosis in control subjects was obstructive sleep apnea (OSA). CONCLUSIONS: TLR2, TLR4, TLR9 and CD14 gene SNPs were not more prevalent in otitis-prone children.

Tissue remodeling gene expression in a murine model of chronic rhinosinusitis.

OBJECTIVE/HYPOTHESIS: The matrix metalloproteinase (MMP), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families regulate tissue remodeling in many normal and pathophysiologic processes. We hypothesize that induction of chronic sinonasal inflammation will be associated with changes in regulation of these tissue remodeling cytokines. METHODS: Balb/c mice aged 8 to 12 weeks were sensitized and treated with intranasal Aspergillus fumigatis (AF) three times per week for 1 week, 3 weeks, 2 months, and 3 months (n = 8 each time point). Sinonasal tissues were evaluated for changes in MMP, FGF, and BMP regulation using standard RT-PCR techniques. Additional snouts were processed for histology and immunohistochemistry. Untreated mouse snouts of identical age were used as controls. RESULTS: Significant upregulation of MMP8 was observed at 2 months, and MMP1a, MMP7, MMP8, and MMP12 were all significantly upregulated at 3 months. FGF3 was significantly upregulated at 3 weeks and 3 months, and FGF5, FGF6, and FGF8 were all significantly upregulated at 3 months. BMP8b and BMP9 were significantly upregulated at 3 months. Histologic analysis revealed mucosal, stromal, and mucin gland hypertrophy, increased mucin production, and metaplasia with loss of cilia. Antibody staining was strongly positive in the AF-treated group. CONCLUSIONS: Induction of CRS is associated with time-dependent changes in tissue remodeling cytokine expression occurring in conjuction with inflammatory tissue changes. Antibody staining for upregulated cytokines suggests local production within the sinonasal mucosa. Further study is required to better understand the association between BMP, FGF, and MMP regulation and tissue remodeling changes resulting from chronic inflammation.

Vascular Pathophysiology in Hearing Disorders.

The inner ear vasculature is responsible for maintenance of the blood-labyrinth barrier, transport of systemic hormones for ion homeostasis, and supplying nutrients for metabolic functions. Unfortunately, these blood vessels also expose the ear to circulating inflammatory factors resulting from systemic diseases. Thus, while the inner ear blood vessels are critical for normal function, they also are facilitating pathologic mechanisms that result in hearing and vestibular dysfunction. In spite of these numerous critical roles of inner ear vasculature, little is known of its normal homeostatic functions and how these are compromised in disease. The objective of this review is to discuss the current concepts of vascular biology, how blood vessels naturally respond to circulating inflammatory factors, and how such mechanisms of vascular pathophysiology may cause hearing loss.

Altered expression of tissue remodeling genes in a mouse model of acute allergic rhinitis.

BACKGROUND: Osteogenesis, fibrosis, and scarring are prominent pathologic changes resulting from chronic sinonasal inflammation, and these tissue changes may increase the degree of disease symptomatology and the level of surgical difficulty. Members of the bone morphogenetic protein (BMP) and fibroblast growth factor (FGF) families of cytokines and the matrix metalloproteinase (MMP) family of endopeptidases are known to regulate tissue remodeling in other disease processes, but their role in acute and chronic sinonasal inflammation remains undefined. METHODS: A previously described mouse model of acute allergic rhinitis secondary to Aspergillus fumigatis exposure in BALB/C mice was used. Intranasal challenge was performed 1 week following intraperitoneal sensitization with A. fumigatis extract and mice were sacrificed 6 hours (n = 8) and 24 hours (n = 8) later. Additional mice were intranasally challenged 3 times per week and sacrificed at the end of 7 days (n = 8) and 21 days (n = 8). The snouts were processed for quantitative reverse-transcription polymerase chain reaction (RT-PCR) and compared to untreated controls for messenger ribonucleic acid (mRNA) expression of BMP1, 2, 3, 4, 5, 6, 7, 8a, 8b, 9, 10, FGF1, 2, 3, 4, 5, 6, 7, 8, 10, and MMP1a, 2, 3, 7, 8, 9, 12, and 14. Additional 21-day-old mice were prepared for sinonasal histopathology. Control mice were treated with the same protocol, with intraperitoneal phosphate-buffered saline (PBS) and intranasal PBS substituted for A. fumigatis extract. Untreated mice were used for additional comparison. RESULTS: Compared to both the PBS-treated and untreated control groups, statistically significant (p < 0.05) upregulation of MMP8 was observed in the 6-hour time point. Significant downregulation of MMP8 was observed at 1 week. Significant upregulation of FGF3 was observed at 1 week (p < 0.05). BMP3 and BMP5 were significantly downregulated in the 1-week group (p < 0.05). The mice exhibited histologic sinonasal changes consistent with allergic inflammation. CONCLUSION: Intranasal exposure to A. fumigatis results in altered expression of several tissue remodeling cytokines at varying time points in the acute allergic rhinitis mouse model. These changes in cytokine regulation may subsequently contribute to sinonasal osteogenesis, scarring, and fibrosis as seen in chronic rhinosinusitis.

Tissue remodeling in the acute otitis media mouse model.

OBJECTIVES: Otitis media is an infectious, inflammatory process involving the middle ear space. Chronic inflammation is associated with fibrosis, scarring and osteogenesis within the middle ear, which may contribute to subsequent hearing loss and increase the difficulty of treatment. METHODS: Heat-killed Streptococcus pneumoniae was injected into the middle ears of 8-12 week old Balb/c mice. Control mice were treated with PBS middle ear injections. Middle ears were harvested at 1, 3, 5 and 7 days following injection (n=8 for each time point). The middle ears were processed using standard RT-PCR techniques. Up- and down-regulation of mRNA expression of various members of the Bone Morphogenetic Protein (BMP), Fibroblast Growth Factor (FGF) and Matrix Metalloproteinase (MMP) families was quantified and compared to PBS treated controls (n=8 for each time point). RESULTS: Significant upregulation of MMP2, MMP3 and MMP9 was observed at varying time points (p<0.05). Significant downregulation of BMP3, BMP4, BMP5 BMP6 and BMP8a was seen at varying time points (p<0.05). Significant downregulation of FGF3, FGF6, FGF10 and FGFr1 was observed at varying time points (p<0.05). No significant expression of BMP8b, BMP9, BMP10, FGF5, FGF8, MMP1a, MMP7 and MMP14 was detected within the middle ear. CONCLUSIONS: Inflammation within the middle ear following injection of bacterial products results in changes in the regulation of several tissue remodeling cytokines and proteinases in the mouse model. Further understanding of these molecular processes may allow for the development of treatment modalities aimed at preventing middle ear tissue remodeling.

Murine middle ear inflammation and ion homeostasis gene expression.

HYPOTHESIS: Ion homeostasis genes are responsible for the movement of ions and water in the epithelium of the middle ear. BACKGROUND: It is not well known to what extent disruption of ion homeostasis is a factor in the accumulation of middle ear fluid during otitis media. METHODS: Balb/c mice were transtympanically injected with heat-killed Hemophilus influenza bacteria. Untreated and saline-injected mice were used as controls. Mice were euthanized at 6, 24, and 72 hours and 1 week after injection, the bullae harvested, and total ribonucleic acid isolated from the middle ear tissues. Ion homeostasis genes were analyzed with real-time quantitative reverse transcription-polymerase chain reaction from the following gene families: Na,K-ATPase, claudins, K transport channels, epithelial Na channels, gap junctions, and aquaporins. Inflammatory genes also were analyzed to document inflammation. RESULTS: All inflammatory genes analyzed were significantly upregulated, more at 6 hours than at 24 hours, with the exception of vascular endothelial growth factor and Mapk8. Most middle ear ion homeostasis genes experienced downregulation because of inflammation. This was most prominent in the aquaporin and Na,K-ATPase genes. Significant upregulation was seen in several genes in response to inflammation and saline independently. CONCLUSION: The innate immune response to bacteria in the middle ear induces expression of several inflammatory genes. Coinciding with this inflammation is the downregulation of numerous ion homeostasis genes that are involved in ion and water transport and maintenance of tight junctions. This may explain the fluid accumulation within the middle ear seen with both acute and chronic otitis media.

Altered expression of middle and inner ear cytokines in mouse otitis media.

OBJECTIVES/HYPOTHESIS: The inner ear is at risk for sensorineural hearing loss in both acute and chronic otitis media (OM), but the mechanisms underlying sensorineural hearing loss are unknown. Previous gene expression array studies have shown that cytokine genes might be upregulated in the cochleas of mice with acute and chronic OM. This finding implies that the inner ear could manifest a direct inflammatory response to OM that may cause sensorineural damage. Therefore, to better understand inner ear cytokine gene expression during OM, quantitative real-time polymerase chain reaction and immunohistochemistry were used in mouse models to evaluate middle and inner ear inflammatory and remodeling cytokines. STUDY DESIGN: Basic science experiment. METHODS: An acute OM model was created in Balb/c mice by a transtympanic injection of Streptococcus pneumoniae in one ear; the other ear was used as a control. C3H/HeJ mice were screened for unilateral chronic OM, with the noninfected ear serving as a control. RESULTS: Both acute and chronic OM caused both the middle ear and inner tissues in these two mouse models to overexpress numerous cytokine genes related to tissue remodeling (tumor necrosis factor-α, bone morphogenetic proteins, fibroblast growth factors) and angiogenesis (vascular endothelial growth factor), as well as inflammatory cell proliferation (interleukin [IL]-1α,ß, IL-2, IL-6). Immunohistochemistry confirmed that both the middle ear and inner ear tissues expressed these cytokines. CONCLUSIONS: Cochlear tissues are capable of expressing cytokine mRNA that contributes to the inflammation and remodeling that occur in association with middle ear disease. This provides a potential molecular basis for the transient and permanent sensorineural hearing loss often reported with acute and chronic OM.

Simultaneous measurement of multiple ear proteins with multiplex ELISA assays.

A recent advancement in enzyme-linked immunosorbent assay (ELISA) technology is the multiplex antibody array that measures multiple proteins simultaneously within a single sample. This allows reduction in sample volume, time, labor, and material costs, while increasing sensitivity over single ELISA. Current multiplex platforms include planar-based systems using microplates or slides, or bead-based suspension assay with microspheres. To determine the applicability of this technology for ear research, we used 3 different multiplex ELISA-based immunoassay arrays from 4 different companies to measure cytokine levels in mouse middle and inner ear tissue lysate extracts 24 h following transtympanic Haemophilus influenzae inoculation. Middle and inner ear tissue lysates were analyzed using testing services from Quansys Biosciences, Aushon Biosystems SearchLight (both microplate-based), MILLIPLEX MAP Sample (bead-based), and a RayBiotech, Inc (slide-based) kit. Samples were assayed in duplicate or triplicate. Results were compared to determine their relative sensitivity and reliability for measures of cytokines related to inflammation. The cytokine pg/ml amounts varied among the multiplex assays, so a comparison also was made of the mean fold increase in cytokines from untreated controls. Several cytokines and chemokines were elevated, the extent dependent upon the assay sensitivity. Those most significantly elevated were IL-1α, IL-1ß, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1ß, IL-6) to assess sensitivity over the traditional method. Overall, the Quansys Biosciences and SearchLight arrays showed the greatest sensitivity, both employing the same multiplex methodology of a spotted array within a microplate well with chemiluminescent detection. They also were more sensitive than the traditional single ELISA performed with commercial kits and matched gene expression changes determined by quantitative RT-PCR. The Quansys array showed a limit of detection for ear IL-6 down to 2-4 pg/ml, indicating it is sufficiently sensitive to detect ear proteins present in low concentrations. Thus, the multiplex ELISA procedures appear suitable and reliable for the study of hearing related proteins, providing accurate, quantitative, reproducible results with considerable improvement in sensitivity and economy.

Low dose combination steroids control autoimmune mouse hearing loss.

The severe side effects of glucocorticoids prevent long term management of hearing loss. Alternative steroid treatments that minimize or eliminate these effects would significantly benefit therapeutic control of hearing disorders. A steroid treatment study of autoimmune mouse hearing loss was conducted to determine the efficacy of combining aldosterone and prednisolone at low doses. An assessment also was made of low dose fludrocortisone, a synthetic mineralocorticoid that also has a slight glucocorticoid effect. MRL/MpJ-Fas(lpr) mice were tested for baseline ABR thresholds at 3 months of age and then treated with aldosterone (3.0 µg/kg) or prednisolone (1.0 mg/kg) to determine the lowest effective dose of each. Other mice were given the two steroids in combination at doses of Pred 0.5 mg+Aldo 1.5 µg; Pred 1.0 mg+Aldo 3.0 µg; or Pred 1.5 mg+Aldo 5.0 µg. Mice were retested with ABR at 1 and 2 months to determine the efficacy of the different steroid treatments in controlling hearing loss. Another series of mice were given the synthetic mineralocorticoid fludrocortisone at low (2.8 µg/kg) or high (10 µg/kg) doses and retested at monthly intervals for 3 months. Autoimmune mouse hearing loss developed in untreated controls. This threshold elevation was not prevented by prednisolone at 1 mg/kg or by aldosterone at 3 µg/kg when each was given alone. However, the two steroids combined at these doses effectively controlled hearing loss. The fludrocortisone treatments also were effective at low doses in preventing or reversing the autoimmune mouse hearing loss. This efficacy of combined steroids at low doses suggests the potential for reducing the side effects of glucocorticoids in the therapeutic control of hearing disorders.