2.1 Establishment of animal model
The OME animal model was established according to a previous study by Hardy et al. [10], which were modified slightly, as follows:
Model group (OME Group) was intraperitoneally injected with 10mg Ovalbumin (Sigma-Aldrich, US) which was dissolved in 0.8ml PBS phosphate buffer (0.01M, pH7.4) plus 5.14mg aluminum hydroxide gel adjuvant, once a week for 2 weeks. 2 weeks after receiving chloramine (100 mg/ml) + stability (5 mg/ml) by intraperitoneal injection of 2:1 mixture (3 ml/kg) depth of anesthesia, the supine position, under the operating microscope is equal to the level of hyoid, a crosscutting was done for separation of hyoid scapula muscle and hyoid bone muscle to expose double ear, bubble bottom wall respectively, aseptic technique was used to trace syringe. OVA 2 mg of 50 uL PBS liquid was injected into bilateral listen to bubble, which is ear stimulating stage (before injection, about 2 mm hole was made from the injection site to balance the middle ear cavity injection pressure and prevents the tympanic membrane rupture). Gelatine sponge was inserted to prolong OVA duration. After injection, 2 small holes were sealed with bone wax, which was the intraaural stimulation stage. Control group (CON) was sensitized with PBS. Intramuscular injection of penicillin prevented bacterial infection before stimulation. After the operation, animals were sacrificed 2 days after feeding in cages in a clean environment. The researchers did not know the allocation of groups when evaluating the results. All animals were treated in accordance with the guidelines for the use of experimental animals which were approved by the Ethnic Committee of Shenzhen Longgang E.N.T Hospital (Shenzhen Otorhinolaryngology Institute).