The Etiology Of Experimental Tympanosclerosis In The Guinea Pig

BackgroundTympanosclerosis is an abnormal condition of the middle ear caused by calcareous deposits in the tympanic membrane, tympanic cavity and occasionally in the mastoid, which is one of the important causes of conductive deafness. The main pathological changes are transparent inherent variability and calcification at the eardrum and middle ear submucosa layer. In 1869, Von Tr(o|¨)ltsch firstly gave a comprehensive description of Tympanosclerosis and forwarded the word "sclerosis". Until 1956 Z(o|¨)llner conducted large of drum shaped operation, and published thesis of Tympanosclerosis, the illness was formally named. The incidence of Tympanosclerosis is different, 20% to 43% oversee, while 3.7% to 11.7% in China. With the development of drum shaped operation and ear microsurgery, Tympanosclerosis is used to otology physician. At present, the clinical treatment of tympanosclerosis is surgery, but the tympanosclerosis lesions often can't be completely removed and formed postoperative scar and conglutination, affecting hearing level. We can see that the treatment of tympanosclerosis is comparatively more complicated and difficult. Surgical treatment causes the patients both mental and physical loss, and the long-term effect still need to be verified, so we look forward to prevent and intervene on the pathogeny, which will reduce the suffering of patients, as well as save health care resources, which is in line with modern medicine requirements. Most scholars believe that the chronic suppurative otitis media and secretory otitis media myringotomy and tube insertion can cause tympanosclerosis, but the specific etiology is unknown. The main etiology of the disease is chronic suppurative otitis media, secretory otitis media myringotomy and tube insertion, the middle ear trauma and bleeding, autoimmune, increased calcium and other factors are related. From the point of evidence-based medicine, we create animal models, to investigate the important etiology of tympanosclerosis.We chose guinea pigs for the experiment, as the anatomical structure of the middle ear of guinea pigs were similar to those of human, so it's easy to operate and observe, as well, with reference to foreign literature, guinea pigs often are chose as scientific ear models.This study was to prove chronic suppurative otitis media, secretory otitis media myringotomy and tube insertions are the main pathogeny for tympanosclerosis, though the guinea pigs model. This study was to set up a model of tympanosclerosis in guinea pig by two methods, one is inoculating 1×10⁸·L^-1 Staphylococcosis aureus into the middle ear cavities, the other is myringotomy and tube insertion. To investigate the structural features of the normal guinea pigs and guinea pigs modeling of the middle ear mucosa in light microscope and electron microscope. At present the tympanosclerosis study in domestic is still less, there are a lot of research spaces and has important clinical value. At the same time, this experiment involves ears microsurgical operation, the surgical skills training and the use of microscopy techniques, which is good for the researcher's ear microsurgery foundation.ObjectiveFrom the point of evidence-based medicine, we create animal models, to investigate and prove the chronic suppurative otitis media and tube insertion is the main cause of tympanosclerosis, observe the development and process of tympanosclerosis, to investigate the structural features of the normal guinea pigs and guinea pigs modeling of the middle ear mucosa in light microscope and electron microscope.Methods1. MaterialsSixty healthy mottle and black-eye guinea pigs, ordinary class, of either gender, weight ranging from 250 to 350g, provided by Animal Experiment Center of Zhujiang Hospital, Southern Medical University. All the guinea pigs were selected with sensitive auricles, entire tympanic membrane, excluding external auditory canal, such as middle ear infections.2. Process of ExperimentThe experiment is parted into three processes:2.1 Model oneThirty healthy guinea pigs were selected as self-control group. After general anesthesia, by inoculating 1×10⁸·L^-1 Staphylococcosis aureus into the middle ear cavities of left ears as experimental group, with the right ears as controls. The supperrative otitis media models were established. Then the animals were sacrificed by decapitation after 1, 3 and 6 months. The histological analysis of middle ear mucosa was performed.2.2 Model two:Thirty healthy guinea pigs were selected as research object. After general anesthesia, only left ears were modeled by myringotomy and tube insertion as experimental group, allowing the contra lateral tympanic membrane and middle ear to serve as controls. After 1, 3 and 6 months, the animals were sacrificed by decapitation to observe eardrum and middle ear mucosa morphology changes. The histological analysis of middle ear mucosa was performed whether there is asclerosis.2.3 Light and electron microscopic analysis was performed on the middle earsubmucosa of the guinea pigs either normal or model.2.3.1 Specimens were divided into two groups: one group of specimens was fixed and stained for light microscopic study with hematoxylin-eosin,2.3.2 The other group of specimens: when the issue was dissected out. it was fixed in phosphate-buffered 2.5 percent glutaraldehyde and post-fixed with 2 percent cosmic acid. After dehydration in a series of graded alcohol, the specimens were embedded in Epon and sectioned. Section were stained with uranyl acetate and studied in the electron microscope. Specific procedure: 1 Tissue dissected out and placed in fixing solution. 2 After washing, the tissue is dehydrated by placing it in higher and higher concentrations of alcohol, and then it is placed in dilute solution of plastic embedding medium Epon. 4 Tissue is placed in final embedding mixture and the plastic is polymerized in an oven. When the plastic is hard, the block is trimmed and is ready for sectioning. Sections are cut on an ultramicrotome with a glass or diamond knife. The sections are floated off the edge of the knife onto the surface of a water trough and then they are picked off the surface with a copper grid. 5 Sections dry. 6After the sections dry, they are ready for staining with heavy metal solutions uranyl acetate and viewing in the electron microscope.Results1. Model one:We consider the perforation of tympanic membrane and liquor puris accumulated in tympanic cavity as the standard of suppurative otitis media, all were modeled successfully.One month after inoculating Staphylococcosis aureus, the experimental group (N=10) appeared normal. There months later, three of the experimental group specimens (N=10) showed calcification. Six months later, five of the experimental group specimens (N=10) showed calcification. The control group showed no calcification. Through statistical analysis, the incidence rate of tympanosclerosis in experimental group (chronic suppurative otitis media) is higher than that of control group. Histological changes consistent with tympanosclerosis were seen in some of animals after 3 months and longer time after the operation. The extent of calcium deposition and fibrosis across the mucosa was related to the duration of supperrative otitis media.2. Model two:One month after eustachian tube obstruction the experimental group (N=9) appeared normal, except that one was to be rejected because of infection. There months later, three of the experimental group specimens (N=10) showed calcification. Six months later, four of the experimental group specimens (N=10) showed calcification. Through statistical analysis, the incidence rate of tympanosclerosis in experimental group (chronic suppurative otitis media) is higher than that of control group. The control group showed no calcification. Histological changes consistent with tympanosclerosis were seen in some of animals after 3 months and longer time after the operation. The extent of calcium deposition and fibrosis across the mucosa was related to the duration of indwelling tubes.3. Morphology:Under microscope: the tympanic membrane of normal guinea pigs is smooth and translucent, the tympanic cavity is thin, smooth, with Pale Pink,. The bone of acoustic capsule is thin and smooth. Model guinea pigs showed different stages of thickness membrane mucosa.Under Light microscope: the mucous membrane of normal guinea pigs tympanic membrane appears thin, the structure is in good order, and without calcification. Model guinea pigs showed varying degrees of mucosal thickening, fibrosis and calcification.Under Electron microscope: normal guinea pigs showed many attic mucosa cells and little collagen fibers. Model guinea pig collagen fibers increased, fibrocytes were elongated and degenerating, lysosome was remarkable, lysosomes were clustered, endoplasmic reticulum expand to sacculation., with calcareous deposits, around collagen fibers and microfibrils.Conclusion1. Tympanosclerosis is a progressive disorder. Our experiment shows that tympanosclerosis can appear in a certain number of cases by inoculating Staphylococcosis aureus into the middle ear cavities of guinea pigs. We propose that this guinea pig model for supperrative otitis media is therefore a suitable animal model for the study of tympanosclerosis. Chronic suppurative otitis media is an important etiology of tympanosclerosis.2. This new guinea pig model for myringotomy and tube insertion is a suitable animal model for the study of tympanosclerosis. Myringotomy and tube insertion is an important etiology of tympanosclerosis.3. Tympanosclerosis of guinea pig performance of high-density fibrosis, glass-like degeneration and calcification in middle ear mucosa. Electron microscopy revealed collagen fibers further significant proliferation and rich electronic calcinosis, changes in the fibroblast-like cells, there are lysosome and mitochondrial degeneration and calcareous deposition.