Optimizing Auditory Brainstem Response Acquisition Using Interleaved Frequencies.

Auditory brainstem responses (ABRs) require averaging responses to hundreds or thousands of repetitions of a stimulus (e.g., tone pip) to obtain a measurable evoked response at the scalp. Fast repetition rates lead to changes in ABR amplitude and latency due to adaptation. To minimize the effect of adaptation, stimulus rates are sometimes as low as 10 to 13.3 stimuli per second, requiring long acquisition times. The trade-off between reducing acquisition time and minimizing the effect of adaptation on ABRs is an especially important consideration for studies of cochlear synaptopathy, which use the amplitude of short latency responses (wave 1) to assess auditory nerve survival. It has been proposed that adaptation during ABR acquisition can be reduced by interleaving tones at different frequencies, rather than testing each frequency serially. With careful ordering of frequencies and levels in the stimulus train, adaptation in the auditory nerve can be minimized, thereby permitting an increase in the rate at which tone bursts are presented. However, widespread adoption of this stimulus design has been hindered by lack of available software. Here, we develop and validate an interleaved stimulus design to optimize the rate of ABR measurement while minimizing adaptation. We implement this method in an open-source data acquisition software tool that permits either serial or interleaved ABR measurements. The open-source software library, psiexperiment, is compatible with widely used ABR hardware. Consistent with previous studies, careful design of an interleaved stimulus train can reduce ABR acquisition time by more than half, with minimal effect on ABR thresholds and wave 1 latency, while improving measures of wave 1 amplitude.

Fetal antisense oligonucleotide therapy for congenital deafness and vestibular dysfunction.

Disabling hearing loss impacts ∼466 million individuals worldwide with 34 million children affected. Gene and pharmacotherapeutic strategies to rescue auditory function in mouse models of human deafness are most effective when administered before hearing onset, after which therapeutic efficacy is significantly diminished or lost. We hypothesize that preemptive correction of a mutation in the fetal inner ear prior to maturation of the sensory epithelium will optimally restore sensory function. We previously demonstrated that transuterine microinjection of a splice-switching antisense oligonucleotide (ASO) into the amniotic cavity immediately surrounding the embryo on embryonic day 13-13.5 (E13-13.5) corrected pre-mRNA splicing in the juvenile Usher syndrome type 1c (Ush1c) mouse mutant. Here, we show that this strategy only marginally rescues hearing and partially rescues vestibular function. To improve therapeutic outcomes, we microinjected ASO directly into the E12.5 inner ear. A single intra-otic dose of ASO corrects harmonin RNA splicing, restores harmonin protein expression in sensory hair cell bundles, prevents hair cell loss, improves hearing sensitivity, and ameliorates vestibular dysfunction. Improvements in auditory and vestibular function were sustained well into adulthood. Our results demonstrate that an ASO pharmacotherapeutic administered to a developing organ system in utero preemptively corrects pre-mRNA splicing to abrogate the disease phenotype.

A neonatal nonhuman primate model of gestational Zika virus infection with evidence of microencephaly, seizures and cardiomyopathy.

Zika virus infection during pregnancy is associated with miscarriage and with a broad spectrum of fetal and neonatal developmental abnormalities collectively known as congenital Zika syndrome (CZS). Symptomology of CZS includes malformations of the brain and skull, neurodevelopmental delay, seizures, joint contractures, hearing loss and visual impairment. Previous studies of Zika virus in pregnant rhesus macaques (Macaca mulatta) have described injury to the developing fetus and pregnancy loss, but neonatal outcomes following fetal Zika virus exposure have yet to be characterized in nonhuman primates. Herein we describe the presentation of rhesus macaque neonates with a spectrum of clinical outcomes, including one infant with CZS-like symptoms including cardiomyopathy, motor delay and seizure activity following maternal infection with Zika virus during the first trimester of pregnancy. Further characterization of this neonatal nonhuman primate model of gestational Zika virus infection will provide opportunities to evaluate the efficacy of pre- and postnatal therapeutics for gestational Zika virus infection and CZS.

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.

Gene Therapy in Mouse Models of Deafness and Balance Dysfunction.

Therapeutic strategies to restore hearing and balance in mouse models of inner ear disease aim to rescue sensory function by gene replacement, augmentation, knock down or knock out. Modalities to achieve therapeutic effects have utilized virus-mediated transfer of wild type genes and small interfering ribonucleic acids; systemic and focal administration of antisense oligonucleotides (ASO) and designer small molecules; and lipid-mediated transfer of Cas 9 ribonucleoprotein (RNP) complexes. This work has established that gene or drug administration to the structurally and functionally immature, early neonatal mouse inner ear prior to hearing onset is a prerequisite for the most robust therapeutic responses. These observations may have significant implications for translating mouse inner ear gene therapies to patients. The human fetus hears by gestational week 19, suggesting that a corollary window of therapeutic efficacy closes early in the second trimester of pregnancy. We hypothesize that fetal therapeutics deployed prior to hearing onset may be the most effective approach to preemptively manage genetic mutations that cause deafness and vestibular dysfunction. We assert that gene therapy studies in higher vertebrate model systems with fetal hearing onset and a comparable acoustic range and sensitivity to that of humans are an essential step to safely and effectively translate murine gene therapies to the clinic.

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.

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.

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.

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.

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.

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.

Steroid control of acute middle ear inflammation in a mouse model.

OBJECTIVE: To investigate steroids for their potential for therapeutic approaches to control otitis media. Glucocorticoids and mineralocorticoids have differential effects on inflammation and fluid absorption, but little is known of their control of middle and inner ear manifestations of acute otitis media. DESIGN: Both glucocorticoid (prednisolone and dexamethasone) and mineralocorticoid (aldosterone and fludrocortisone) steroids were investigated for their ability to reduce inflammatory symptoms in a mouse otitis media model. SETTING: Academic medical center. SUBJECTS: Acute inflammation was induced by transtympanic injection of heat killed Streptococcus pneumoniae to 100 BALB/c mice. INTERVENTIONS: Twenty mice in each experimental group (prednisolone, dexamethasone, aldosterone, and fludrocortisone) were given a steroid in their drinking water the day before inoculation, and these treatments were continued until the mice were killed for histologic examination. Twenty control mice were treated with water only. MAIN OUTCOME MEASURES: Histologic measure of inflammation: middle ear fluid, inflammatory cell number, and tympanic membrane thickness. RESULTS: Histologic middle ear morphometrics showed significant steroid effects at both 3 and 5 days in reduction of fluid area, cell number, and tympanic membrane thickness. CONCLUSIONS: Glucocorticoids were most effective in controlling inflammation. Interestingly, the mineralocorticoids were also effective in reducing the inflammatory response at 5 days, suggesting that their fluid transport function helped clear disease. Thus, steroid control of middle ear disease may be useful in alleviating symptoms faster and reducing the risk to the inner ear.

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.

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.

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.

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.