Mouse Middle Ear Ion Homeostasis Channels and Intercellular Junctions

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.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.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.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.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

Aquaporins.

<p>Aquaporins 1, 4, and 5 in the inner ear (A-C) and middle ear (D-G). A: AQP1 in the inner ear showed positive staining of the lateral lining of the spiral ligament and some fibrocytes within the spiral ligament. B: AQP4 in the inner ear positively stained bone of the otic capsule, organ of Corti, and spiral ganglion. C: AQP5 showed mild labeling of the inner ear organ of Corti. D: AQP 1 antibody stained the middle ear epithelial submucosa and capillary endothelium. E and F: Middle ear AQP4 labeled the middle ear epithelium itself and portions of the ossicular bones, malleus and incus. G: AQP5 in the middle ear appeared limited to the apical membranes of the epithelium. Negative controls are displayed as inset pictures. [SV, stria vascularis; SM, scala media; SL, spiral ligament; RM, Reissner’s membrane; OC, organ of Corti; ST scala tympani; MEE, middle ear epithelium; SUB, submucosa; MES, middle ear space; M, malleus; I, incus].</p

Ion Channels.

<p>A: ENaC in the inner ear occurred in the apical membrane of the crista ampullaris, as well as other places. B: Na+,K+-ATPase staining in the inner ear stained the stria vascularis, fibrocytes of the spiral ligament, and the organ of Corti. C: ENaC in the middle ear was located in the apical membrane of the epithelium. D: Na⁺,K⁺-ATPase middle ear diffusely stained the lining epithelium. E: KCNQ1 of the inner ear occurred at the apical surface of marginal cells of stria vascularis. F: KCNJ10 in the inner ear was seen, staining the intermediate cells of stria vascularis and organ of Corti. G: KCNQ1 stained the middle ear epithelium. H: KCNJ10 also stained the middle ear epithelium. Negative controls are displayed as inset pictures. [CA, crista ampullaris; SV, stria vascularis; SM, scala media; SL, spiral limbus; RM, Reissner’s membrane; OC, organ of Corti; MEE, middle ear epithelium; MES, middle ear space].</p

Comparison of Inner Ear and Middle Ear Antibody Staining.

<p>TM: tympanic membrane; ME: middle ear.</p

Primary and Secondary Antibodies.

<p>Primary and Secondary Antibodies.</p

Junctional Complexes.

<p>A: Claudin 3 in the inner ear localized to the organ of Corti, Reissner’s membrane, periosteal lining of otic capsule, and the apical surface of the marginal cells of stria vascularis. B: GJB2 in the inner ear positively stained between fibrocytes in the spiral ligament, as well as cells within the organ of Corti and spiral limbus. C: Claudin 3 in the middle ear occurred in the intercellular junctions along lateral and basal cell membranes in the mucosal epithelium. D: GJB2 in the middle ear also stained the middle ear epithelium. Negative controls are displayed as inset picture. [SV, stria vascularis; SM, scala media; RM, Reissner’s membrane; OC, organ of Corti; MEE, middle ear epithelium; MES, middle ear space].</p