Immunohistochemical Investigation of Inducible Nitric Oxide Synthase, Osteopontin, and Calcium-Sensing Receptor in a Myringosclerosis/Tympanosclerosis Model

Abstract

To investigate roles of types of inflammation, inducible nitric oxide synthase (iNOS), osteopontin (OPN), and calcium sensing receptor (CaSR) in the tympanic membrane and middle ear in etiopathogenesis of myringosclerosis/tympanosclerosis (MT). Etiopathogenesis of myringosclerosis/tympanosclerosis is still unclear. Clinical and experimental observations demonstrate that hyperoxygenation might induce tympanosclerosis. Seventy-five rats were divided into 3 groups: ventilation tube (VT) insertion, the Eustachian tube (ET) obliteration, and both procedures. Right ears were selected for mentioned interventions. Left ears served as controls. Then, histopathologic and immunohistochemical investigations were performed in tympanic bulla. MT and inflammation in tympanic membrane and middle ear space were investigated. Immunohistochemical investigation included staining with iNOS, OPN, and CaSR. Overall 42.7% of all rats developed MT. There was no significant difference in MT incidence among the groups (ET + VT group: 56%; ET group: 44%; VT group: 28%; p > 0.017). iNOS expression occurred in 30.6% of the intervention groups with insignificant differences (ET + VT group: 40%; ET group:36%; VT group:16%; p > 0.05). There was no significant difference in iNOS expression between tympanosclerotic (25%) and non tympanosclerotic ears (34.9%) (p = 0.359). OPN was expressed in 82.6% overall. It was the highest for ET group and ET + VT group (92% for each) followed by VT group (64%). There was a marginal significance in comparison of OPN staining between VT group and ET group and also between VT group and ET + VT group (p = 0.017). There was a significant difference in OPN expression between tympanosclerotic (100%) and nontympanosclerotic ears (69.8%) (p = 0.001). Neither control ears nor intervention groups showed CaSR expression. Comparisons of inflammation of the tympanic membrane and middle ear space between tympanosclerotic and non-tympanosclerotic ears yielded significant differences (p = 0.003, p = 0.002, respectively). Tympanosclerotic ears had a tendency to show chronic or mixed inflammation in contrast to non-tympanosclerotic ears (p < 0.017). Filled-middle ear space was seen in 25% of the intervention groups with no significant difference (p > 0.017). There was a significant difference in the incidence between tympanosclerotic (46.8%) and non-tympanosclerotic ears (7%) (p < 0.017). Based on these findings, iNOS may not be evident in stage of MT. OPN staining is strongly associated with the development of MT. CaSR has no role in formation of MT. The results proved roles of mixed or chronic inflammation and the presence of the filled-middle ear in development of MT.