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Ion homeostasis genes are responsible for the movement of ions and water in the epithelium of the middle ear.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.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.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.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.