J. Beth Kempton

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.

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.

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.

Failure of elevated heat shock protein 70 antibodies to alter cochlear function in mice

Heat shock protein 70 (HSP70) has been suggested as the putative cochlear antigen underlying a proposed autoimmune etiology in certain cases of Menieres disease and idiopathic hearing loss. To determine if antibodies to this cellular protein are capable of altering cochlear function, BALB/c (N= 3) and CBA/J (N= 9) mice were inoculated with bovine HSP70 by intraperitoneal injections (10 microg in saline) every 10 days for 7 or 10 months, respectively. An equal number of control mice were injected with PBS according to the same schedule. ABR thresholds at 4, 8, 16, and 32 kHz in the HSP70-inoculated mice did not change over the 10 month period and were similar to saline controls. Furthermore, serum immune complexes and antinuclear antibodies did not increase over the inoculation period. ELISA analysis demonstrated the mice created antibodies to the foreign HSP70, but these apparently caused no abnormalities in the auditory or immune systems. It was concluded that foreign HSP70 is antigenic and inoculation with it will raise antibodies, but these antibodies were neither immunopathogenic nor cochleopathic. Therefore, these findings do not support current theories that elevated anti-HSP70 antibodies are the underlying cause of hearing loss in patients with such antibodies present.

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.

Aldosterone and prednisolone control of cochlear function in MRL/MpJ-Faslpr autoimmune mice

Recently this laboratory showed aldosterone, a mineralocorticoid that only enhances sodium transport, was as effective as the glucocorticoid prednisolone in restoring cochlear function in autoimmune mice. To further test this relationship between sodium transport and autoimmune hearing loss, dosage comparisons were made of prednisolone and aldosterone control of the auditory dysfunction in autoimmune MRL/MpJ-Fas(lpr) mice. Mice were tested at 2 months of age to establish baseline auditory brainstem response (ABR) thresholds, hematocrit, serum immune complexes, and anti-nuclear antibodies. Mice were then given different doses of prednisolone or aldosterone in their drinking water for 2 months. After the treatment period, most untreated water controls showed elevation of ABR thresholds due to the ongoing autoimmune disease. However, the steroid groups had significantly more mice with improved or unchanged thresholds. Both steroids improved stria vascularis morphology, although aldosterone appeared to be more effective. The immune suppressive prednisolone caused a dose-related improvement in levels of serum immune complexes and hematocrit, hallmarks of systemic autoimmune disease. Aldosterone, which has no immune suppressive function, did not alter systemic disease. The comparable efficacy of prednisolone and aldosterone in restoring auditory function suggests steroid reversal of autoimmune hearing loss in mice is due to increasing stria vascularis sodium transport and not suppression of systemic autoimmune reactions.

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-1β, IL-6, TNFα, VEGF, and IL-8/MIP-2. The results of the multiplex systems were compared with single ELISA kits (IL-1β, 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.

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.

Steroid treatment improves cochlear function in the MRL.MpJ-Faslpr autoimmune mouse

Corticosteroid therapy is used to reverse autoimmune sensorineural hearing loss, although little is known of the mechanism by which this occurs. This has been due to the lack of a suitable animal model with spontaneous hearing loss that is steroid responsive. The present study examined the effects of prednisolone treatment on auditory thresholds in the MRL.MpJ-Fas(lpr) autoimmune mouse to determine its suitability as such a model. Autoimmune mice at 3.5-4. 5 months of age were evaluated by pure-tone auditory brainstem response (ABR) to establish threshold elevations due to the disease. The steroid treatment group was then given prednisolone in their drinking water for 2.5 months, while untreated controls were given tap water. Significantly more steroid treated mice survived to the time of post-treatment ABR evaluation. Half of the steroid treated ears demonstrated either improvement or no change in cochlear function compared to only 25% in the untreated controls. Overall, cochlear thresholds in the untreated controls increased by 14.7 dB, whereas no significant threshold increase was seen in the steroid treated group (4.3 dB) over the treatment period. No qualitative anatomical differences were seen in the ears of those mice surviving to the end of the study. These findings establish the autoimmune mouse as a model for studies of steroid responsive mechanisms within the ear. This could apply to autoimmune sensorineural hearing loss, as well as any hearing disorder for which steroid therapy is recommended.

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

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.