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.