Tissue preparation and histopathological examination
All procedures were conducted under hygienic, but not sterile conditions. The animals were sacrificed after anesthesia and the tympanic membrane and tympanic bulla were removed with microdissection. The specimens were fixed for 24 hours in % 10 formaldehyde solution. Then they were decalcified for one week in a %10 formic acid solution. After the fixation and decalcification procedures the specimens were cut into two by transverse cutting. Then they were dehydrated in baths of dereceli alcohol and tissue tracking procedure was applied, and later they were buried in paraffin. Cross sections with a thickness of 5 microns were taken. All cross sections were dyed with hematoxylin and eosin and then were examined with a light microscope (Zeiss Axiophot Axioplan, Germany) by a single expert pathologist. In the examination, the tympanic membrane and the middle ear mucosa were evaluated according to various pre-determined histopathological properties (presence of inflammatory cells, presence of fibrosis, presence of keratinized epithelium in the middle ear (cholestearoma), thickness of the tympanic membrane, thickness of the tympanic bulla mucosa). The measurements of the thickness of the tympanic membrane and tympanic bulla were taken under a 10x magnifying objective.