Ion Homeostasis Channels in Middle Ear Inflammation

Lisa Morris, MD, Beth Kempton, Jacqueline DeGagne, Francis Hausman, Dennis Trune, PhD, MBA Oregon Health & Science University ABSTRACT INTRODUCTION RESULTS DISCUSSION AND CONCLUSION

Objective: Otitis media is a common disorder affecting approximately 90% of children All channels and junctions stained in the normal middle ear epithelium except for claudin 4 while all stained in the inflamed middle ears. Generally the Fluid and ion homeostasis within the middle ear is controlled by a To elucidate the presence and locations of ion and is the leading cause of conductive hearing loss (CHL) in children inflamed mucosa showed more staining reflecting the proliferation of mucosa and increased secretory cells in response to the bacteria. The localization of series of channels and junctions that are well-described to tightly homeostasis channels and junctional within the worldwide1. For normal hearing to occur, a fluid-free and air-filled middle ear channels remained similar between normal and inflamed ears with all channels and junctions except for 4, claudin 4 and . control fluid and ion composition within the inner ear. While the normal middle ear epithelium of mice and to evaluate changes that occur in response to acute inflammation. cavity is required for effective sound transmission. Fluid accumulation in the mechanism of fluid clearance in the middle ear is poorly understood, middle ear is a sequela of acute otitis media and can persist greater than 3 the distinctive locations of many of these transporters, especially the Methods: months, leading to chronic otitis media. Prolonged middle ear effusion and , suggest that the middle ear fluid is regulated by a Immunohistochemistry was performed on paraffin- resultant CHL can lead to language and developmental delay. Often surgical coordinated process. During acute inflammation of the middle ear embedded middle ear sections of 5 BALB/c mice with intervention is required to eliminate the fluid and correct the hearing loss in mucosa, fluid accumulation occurs within the middle ear cavity. A middle ear inflammation after transtympanic injection persistent effusions. recent study has shown that expression for the ion and water with heat-killed Hemophilus influenza and 5 control channels is either downregulated (AQP 1,5, and Na+,K+-ATPase) or BALB/c mice. Primary antibodies included Na+,K+- ATPase α1, epithelial (ENaC), gap Transepithelial ion and fluid transport is one mechanism by which effusions unchanged (ENaC, GJB2, KCNQ1 and KCNJ10) during acute junction beta 2 (GJB2), aquaporins 1, 4 and 5, are cleared from the middle ear, but is not fully understood. Within the inner inflammation, but the junctional complexes (claudin 3 and 4) are claudin 3 and 4, potassium voltage-gated channel ear, the regulation of fluid and ion movement is essential for normal auditory upregulated5. This indicates that extracellular fluid accumulation may (KCNQ1), and potassium inwardly-rectifying channel and vestibular function2. The same ion and fluid channels active in the inner be due to dysfunction of the transepithelial ion and fluid channels (KCNJ10). Sections were then observed and ear may have similar roles with fluid regulation in the middle ear epithelium. within the middle ear epithelium, tipping the homeostatic balance photographed with a fluorescence microscope and towards middle ear effusion. The upregulation of the tight junctions compared. Preliminary studies have shown the presence of at least 10 ion transport and fluid channels, such as the epithelial sodium channel (ENaC) and aquaporins, (claudin 3 and 4) form water barriers between cells and therefore blocks Results: within the middle ear epithelium and support their active role in fluid egress of fluid out of the middle ear cavity. The most prominent staining ion channels in the normal homeostasis3. Little is known what changes occur to these channels in the middle ear included aquaporins 1, 4 and 5, claudin 3, presence of acute middle ear inflammation. This study is to evaluate the On the protein level, this study demonstrates the presence and varied ENaC and Na+,K+-ATPase. GJB2, KCNJ10 and KCNQ1 expression of these channels and junctions at the protein level during the locations of these channels in the normal middle ear epithelium. It also had only moderate staining, while no staining occurred time of maximum inflammation and compare these findings to normal murine demonstrates that the ion and fluid channels are still present and their with claudin 4. The inflamed middle ear epithelium middle ear epithelium. locations are maintained during acute inflammation. Many of the showed expected cellular hypertrophy, but with no loss of channel expression. ENaC, Na+,K+-ATPase, KCNQ1 changes found during the acute inflammatory phase occurred at the and KCNJ10 showed increased staining of the mucosal level of the tympanic membrane. Interestingly aquaporin 4, claudin 4 β surface of tympanic membrane. Aquaporin 4, claudin 4 Figure 1. Aquaporins (AQP). In the normal ear AQP1 stained very strongly in the submucosa and Figure 2. Junctional Proteins. Claudin 3 stained strongly throughout all middle ear mucosa. There is no and gap junction 2 developed strongly staining inclusion bodies and GJB2 demonstrated abnormal staining of inclusion surrounding capillaries and venules. There was no change to expression or localization in the inflamed change in expression of the location of expression in inflamed mucosa, however the tight junctions are within the mucosal layer of the tympanic membrane. The significance of bodies within the cytoplasm of epithelium on the epithelium. The inflamed tympanic membrane (TM) is thickened and edematous, but AQP1 continues to more abundant as the epithelium proliferates. The tympanic membrane shows reactivity of the mucosal which is unknown. Further study is needed to better understand these mucosal surface of the tympanic membrane. be localized to the submucosa of both the endothelial and epithelial linings. AQP4 was found surface but not the epithelial surface of the TM. Claudin 4 was not found to have significant staining throughout the cytoplasm in the normal middle ear and was more uniform throughout the cytoplasm of compared to negative controls within normal middle ear epithelium. There was strong staining present transport processes which may guide targeted therapy to prevent or METHODS AND MATERIALS the inflamed middle ear epithelium. In the normal TM the epithelial layer was more reactive, but not in of inclusion bodies within normal middle ear mucosa overlying the TM in inflamed ears. Gap Junction Conclusions: treat middle ear effusions, therefore preventing numerous surgeries and the inflamed TM. Here there are abnormal inclusion bodies within the mucosal epithelium. AQP5 was was found only in a small amount in the cytoplasm of the middle ear epithelium of both normal and prolonged conductive hearing loss. These findings show that ion homeostasis channels present on the apical surface of the middle ear mucosa in both normal and inflamed ears. This inflamed ears. In the normal TM it was strongly reactive in the epithelial layer however in the inflamed and junctional proteins are present in the normal middle 10 BALB/c mice were screened for absence of middle ear fluid with localization is consistent on the mucosal surface of both normal and inflamed TMs. Black and white TM it was most reactive in the hypertrophic mucosal layer. ME, middle ear; SUB, submucosa; ECT, ectodermal ear mucosa but are not significantly altered in otomicroscopy. Inflammation was induced in five mice utilizing the acute pictures from confocal microscope. ME, middle ear; SUB, submucosa; ECT, ectodermal layer of TM layer of TM; FIB, fibrous layer of TM expression or localization at the protein level during middle ear disease mouse model described previously4. Acute otitis media acute inflammation. Further elucidating these transport (OM) was created by bilateral transtympanic inoculation with heat-killed processes may allow for improved medical treatment of ANTIBODY INNER EAR NORMAL MIDDLE EAR INFLAMED MIDDLE EAR effusions present after acute otitis media and chronic Hemophilus influenza. These mice were sacrificed 72 hours later after Lining of scala tympani Submucosa of ME epithelium Submucosa of ME epithelium REFERENCES effusions leading to protracted ear disease. confirmation of middle ear fluid and their bullae were harvested for Lining of semicircular canal Submucosa of TM Crista ampullaris Submucosa of TM processing. The remaining five mice did not have any intervention and Type III fibrocytes of spiral ligament Submucosa of round window membrane Submucosa of round window membrane Spiral ganglion ME epithelium, cytoplasm Research supported by NIH-NIDCD R01 DC009455 and Organ of Corti ME epithelium, cytoplasm served as controls with absence of middle ear disease. Aquaporin 4 Bone of otic capsule Mucosal layer of TM, inclusion bodies 1. Tos M. Epidemiology and Natural History of Secretory Otitis. American Bone of ossicles Lining of scala tympani Epithelium on ME surface of round window membrane P30 DC005983. Immunohistochemistry was performed on paraffin-embedded middle ear Lining of scala vestibuli Journal of Otology 1984; 5:459-462. ME epithelium, apical surface ME epithelium, apical Aquaporin 5 Organ of Corti sections with the inner ears serving as controls (not shown). Primary Surface of basal cells Surface of basal cells 2. Lang F, Vallon V, Knipper M, Wangemann P. Functional significance of + + Marginal cells of stria vascularis, apical surface channels and transporters expressed in the inner ear and kidney. Am J antibodies (Table 1) included Na ,K -ATPase α1, epithelial sodium channel Reissner's membrane Reticular membrane ME epithelium, basal and lateral surfaces Claudin 3 Spiral ganglion ME epithelium, basal and lateral surfaces Physiol Cell Physiol 2007;293:C1187-208. (ENaC), beta 2 (GJB2), aquaporins 1, 4 and 5, claudin 3 Organ of Corti Mucosal layer of TM Maculae, apical surface 3. Morris LM, DeGagne JM, Kempton JB, Trune DR. Ion homeostasis channels and 4, and potassium channels (KCNQ1 and KCNJ10). Sections were then Periosteal lining of otic capsule Spiral ganglion No significant staining of ME epithelium in middle ear epithelium. Presented at Association for Research in Claudin 4 No significant staining observed and photographed with a fluorescence or confocal microscope and Organ of Corti Mucosal layer of TM, inclusion bodies Otolaryngology Midwinter Meeting, Anaheim, CA USA, February 6-10, 2010. Organ of Corti Spiral ligament compared. ME epithelium, cytoplasm 4. MacArthur CJ, Hefeneider SH, Kempton JB, Parrish SK, McCoy SL, Trune DR. Spiral limbus ME epithelium, cytoplasm GJB2 Lining of utricle Mucosal layer of TM, inclusion bodies Epithelial Layer of TM Lining of saccule Epithelium covering promontory Evaluation of the mouse model for acute otitis media. Hearing Research, Macula, apical surface Lining of semicircular canals 2006;219:12-23. Organ of Corti Macula, apical surface ME epithelium, cytoplasm 5. MacArthur CJ, Hausman F, Kempton JB, Trune DR. Impact of middle ear ENaC Fibrocytes of spiral ligament ME epithelium, cytoplasm Mucosal and epithelial surface of TM Reissner's membrane Epithelium covering promontory inflammation on ion homeostasis gene expression. Presented at Association Spiral ganglion Stria vascularis for Research in Otolaryngology Midwinter Meeting, Anaheim, CA USA, Organ of Corti ME epithelium, cytoplasm February 6-10, 2010. Na+, K+-ATPase Fibrocytes of spiral ligament (Type II & IV) ME epithelium, cytoplasm Spiral ganglion Mucosal layer of TM Lisa Morris MD Reissner's membrane Marginal cells of stria vascularis ME epithelium, cytoplasm KCNQ1 ME epithelium, cytoplasm Oregon Health and Science University Vestibular dark cells Mucosal and epithelial surface of TM

Figure 3. Ion Channels. Each of these ion transport channels were found to be present diffusely within Intermediate cells of stria vascularis ME epithelium, cytoplasm Email: [email protected] KCNJ10 ME epithelium, cytoplasm the cytoplasm of middle ear mucosa of both normal and inflamed ears. In the inflamed middle ear Organ of Corti Mucosal and epithelial surface TM mucosa ENaC was not as localized to the apical surface as in the normal mucosa. Normal TMs had + + Table 2. Summary of primary antibody locations in the inner ear, normal middle ear and inflamed similar staining, however, ENaC and Na ,K -ATPase localized to the mucosal surface of the TM in the middle ear. inflamed ears. ME, middle ear; ECT, ectodermal layer of TM Table 1. Primary antibodies used for immunohistochemistry on paraffin-embedded mouse middle ear and inner ear tissue.

Poster Design & Printing by Genigraphics® - 800.790.4001