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.

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.

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.

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.