The Regulative Effect Of OPG/RANKL System On The Pathogenesis Of Experimental Tympanosclerosis In Rats

Baekground:Tympanosclerosis (TS) is characterized by hyaline changes of the lamina propria of the tympanic membrane and submucosa of middle ear, which may progress to calcification or ossification and induce progressive conductive deafness. At present, there is no effective treatment method of TS. Surgical operation maybe could improve the hearing of some patients with TS in the short term,but it can not prevent the development of TS and run a poor long-term follow-up result. The pathogenesis of TS is still unclear, which related to otitis media, grommet insertion, disturbance of calcium metabolism, oxygen radical, immunologic mechanisms and hereditary factors, but chronic otitis media is widely thought to be the main etiology of TS. Experimental animal models are widely used to study the diseases of middle ear. Although a number of animal models of TS had been established through different method, but it is still lack of a widely accepted one.The pathologic process of TS is progressive, in which the pathological lesions in tympanum or tympanic mucosa eventually vary from hyaline degeneration to calcification or ossification. Previous studies have shown that a number of immunocompetent cells and cytokines which were secreted from these cells participate in the fibrotic and calcific process of tympanic mucosa after bacterial infection of the middle ear, which play an important role in the development of TS. Recent studies demonstrate that osteoprotegerin(OPG) and receptor activator of NF-KB ligand(RANKL) are the important factors which regulate the dedifferentiation and activity of osteoclast.OPG/RANKL system is the last common pathway to regulate bone metabolism.which play an important role in the process of pathologic bone remodeling. Calcification and ossification are also the pathological changes of TS, but the effect and mechanism of OPG/RANKL system on the development of TS are unknown.Our study was to investigate the regulative effect of OPG/RANKL system on the pathogenesis of experimental TS in Sprague-Dawley (SD) rats through detecting the expression of OPG and RANKL in the pecimens of tympanic mucosa by a immnohistochemical staining technique. If proving that OPG/RANKL system have a decisive effect on the pathogenesis of TS, it could be expected to prevent or reverse the pathological process of TS which would be much helpful to prevent and treat TS.Objective:We aim to establish an experimental model of TS in SD rats by inoculating a streptococcus pneumoniae solution into the middle ear. and investigate the regulative effect of OPG/RANKL system on the pathogenesis of TS in rats.Methods:Our study included two steps, in the first step we established the experimental model of TS in rats.in the second step we stained the pecimens of tympanic mucosa in the rats with hematoxylin eosin (HE) and immunohistochemistry.1.A total of30healthy adult male SD rats were included in this study。The animals were randomly separated into two groups,20rats in model group and10rats in control group, and were performed general anesthesia with intramuscular valium and ketamine.The right ears of rats in model group(a total of20ears) were used to create experimental model of TS by inoculating0.05mL of solution containing of1×108CFU/ml streptococcus pneumonia into the middle ears.and the tright ears of rats in control group(a total of10ears) were dealed with nothing serveing as blank controls.All the animals were observed under otomicroscopy after1week,2weeks,4weeks,6weeks and8weeks to evaluate the degree of vascular reaction of tympanum, empyema in tympanum and TS.2.A11animals were sacrificed sacrificed at the end of8weeks after inoculating following general anesthesia. We collected the tympanic bullaes of right ears of rats in model group which appered TS as TS group and the normal tympanic bullaes of right ears of rats in control group as normal group.All the specimens were fixated in4%paraformaldehyde and then decalcified in10%EDTA。After decalcification, the specimens were embedded in paraffin and sectioned on5μm slices. Finally, the specimens were stained with HE to detect pathological changes and immunohistochemistry to detect expression of OPG and RANKL in tympanic mucosa respectively.Results:1.All the10rats in control group had survived up to eight weeks, none of the10right ears of which had acute suppurative otitis media and tympanosclerosis.Of the20rats In model group,17rats had survived up to eight weeks and3rats died in the first week. Of the17right ears of the remaining rats in model group, all the17ears (100.0%) had acute suppurative otitis after1week and a total of13ears(76.5%) had tympanosclerosis after8weeks. Significant differences was both found when Comparing the degree of vascular reaction of tympanum and empyema in tympanum of rats in model group in different period after inoculating respectively (χ2=46.92, P<0.001; χ2=47.25, P<0.001).The degree of vascular reaction of tympanum and empyema in tympanum of rats kept decreasing along with the increase of experimental time. Significant differences was both found when Comparing the degree and incidence of TS of rats in model group in different period after inoculation respectively (χ2=27.09, P<0.001; χ2=12.49, P<0.001).The degree and incidence of TS of rats kept increasing along with the increase of experimental time.2.A total of13specimens of TS group and a total of10specimens of normal group were collected for HE and immnohistochemical staining。THE result of HE staining of specimens in TS group showed that tympanum and tympanic mucosa had become incrassated and irregular and fibroplasia and hyaline degeneration had been seen in lamina propria of the tympanic membrane and submucosa of middle ear, compared with normal group. THE result of immnohistochemical staining showed that OPG and RANKL were expressed in the specimens of both TS group and normal group.which were located to the cytoplasm of epithelium and fibroblasts with brown colour.The expression level of OPG in tympanic mucosa was3.50±1.49in normal group and4.92±0.94in TS group. The expression level of OPG was significantly higher in TS group than in normal group (t=2.802, P=0.011). The expression level of RANKL in tympanic mucosa was3.00±0.94in normal group and2.68±1.35in TS group.There was no significant difference of the expression level of RANKL between normal group and TS group. The ratio of expression level for OPG and RANKL (the expression level of OPG/the expression level of RANKL) was1.21±0.48in normal group and2.26±1.28in TS group. The ratio of expression level for OPG and RANKL was significantly higher in TS group than in normal group (t=2.452, P=0.023)Conelusion:l.The method for establishing experimental TS in rats by inoculating streptococcus pneumonia into the middle ears was effective. The experimental time had an effect on the formation and development of TS in rats.2. OPG/RANKL system may play an regulative role in the development of TS in rats.The increasing of the the ratio of OPG to RANKL may have relation to the development of TS.