LAUSR

OBJECTIVES Our preliminary study performed on perforated rat's tympanic membrane (TM) using Rat Wound Healing RT2 Profiler PCR Array showed significantly increased levels of mRNA for collagens type I and V. Enhanced expression of those genes does not assure that their protein products are indeed present, and in what quantity. Therefore, this study was undertaken to analyze the collagen type I and V content in the healing TM. METHODS Sixty rats were used, of which 10 served as controls and the others had their TM perforated. The experimental animals were divided into five subgroups on the basis of time points (03, 06, 09, 14, 20 day after injury). Videootoscopy and histology were employed to assess the morphology of the healing process. The expression of collagen type I and V was evaluated using Western blot analysis. Tissue localization of collagens was determined by the immunofluorescence method. RESULTS The collagen type I expression was three times higher on the third day after injury and remained on that level for whole period of observation, up to day 20. The increase of the collagen type V expression was gradual, reaching the highest level on day 14 following injury. In comparison to the control TM statistically significant increase in the level of expression was observed starting from day 09 to the end of observation period. In healing TM immunofluorescent labeling of collagen type I and V was seen on the surface of remnants of previous lamina propria and in the loose proliferating fibrous tissue. On day 20 immunofluorescence was present mainly on the surface of thin connective tissue layers forming the scar in the place of previous perforation. CONCLUSION Although the collagens type I and V are present only in subepithelial layer in the normal rat's TM they play significant role in TM healing process.