ABSTRACT
PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.
Subject(s)
Animals , Mice , Ear, Middle/metabolism , Microscopy, Electron, Scanning , Mucin 5AC/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SpectrophotometryABSTRACT
PURPOSE: There is much confusion surrounding the methods of RNA extraction from the middle ear mucosa of mice. In this study, we worked to develop a "melting method," which is faster, purer, and more reliable than other methods in common use. MATERIALS AND METHODS: Thirty-two ears were used for this study. Light microscopy with hematoxylin-eosin staining of the bullae, scanning electron microscopy (SEM), spectrophotometer analysis, and reverse transcription polymerase chain reaction were performed before and after melting the half lateral bullae, which were detached from the temporal bone by using a lateral retroauricular approach. RESULTS: Each resected half bulla contained a well distributed mucosal membrane. After a TRIzol melting duration of 10-30 minutes, only mucosal marker (MUC5AC) was expressed without bony marker (total osteocalcin). The same results were determined from SEM. CONCLUSION: This melting method, compared with stripping and irrigation methods, is effective and offers an easier, more robust approach to extracting RNA from the middle ear mucosal membranes of mice.
Subject(s)
Animals , Mice , Ear, Middle/metabolism , Microscopy, Electron, Scanning , Mucin 5AC/genetics , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , SpectrophotometryABSTRACT
Objective To explore the expression of transforming growth factor ?1(TGF-?1)in the mucosa of guinea pigs with otitis media with effusion(OME)and the role of TGF-?1 in the development of OME.Methods 70 guinea pigs were randomly devided into 7 groups,each with 10 animals.OME was produced by injecting deactivated streptococcus pneumoniae into the typmpanic cavity of guinea pigs.The expression of TGF-?1 was examined by means of immunohistochemistry 6 h,1 d,3 d,7 d,14 d and 30 d after injection.Results TGF-?1 expression could be detected in the middle ear mucosa at 7 d after injection and reached maximum at 30 d.Conclusion TGF-?1 is expressed in the middle ear mucosa of the guinea pigs with OME induced by deactivated streptococcus pneumoniae,and this indicates that TGF-?1 may play a role in the chronic protraction of OME.
ABSTRACT
BACKGROUND AND OBJECTIVES:Prevention of mucosal changes induced by experimental pneumococcal otitis media with antibiotics was reported in the previous studies. But the effects of combined therapy with antibiotics and steroid on middle ear mucosa in acute otitis media is not determined yet. The purpose of this study was to investigate the treatment effects of antibiotics and steroid on the middle ear mucosa of experimental acute otitis media. MATERIALS AND METHOD: The right middle ear of 27 rats was inoculated with log phase type 3 streptococcus pneumoniae, and the left ear served as control. Penicillin G was administered to 9 rats, penicillin G and dexamethasone were administered to 9 rats after bacterial challenge. The animals were killed at day 4, 7 or 14 after challenge. Tympanic membrane and middle ear mucosa were examined under the surgical microscope and light microscope. RESULTS: Untreated pneumococcal AOM has been shown to cause persistent structural changes of the middle ear mucosa. Both in antibiotics-treated group and in antibiotics and steroid-treated group, the structural changes were diminished, as compared with those of untreated infected controls. The antibiotics and steroid-treated group showed marked decrease of structural changes, especially in mucosal metaplasia to secretory epithelium. CONCLUSION: The results suggest that the combination treatment of antibiotics and steroid in acute otitis media would seem to be almost as effective in reducing treatment period and preventing the persistence of mucosal changes, which may decrease development of serous otitis media as a sequale of acute otitis media.