动物模型,内耳病 z-bo 2020-08-08 474

　　(1) Reproduction method Take 4 weeks old inbred male C57BL/6 mice, and guinea pigs weighing 300-400g, with normal ear reflex and no middle ear infection. After the guinea pigs were anesthetized, the heart was perfused, the temporal bones were decapitated, and all inner ear membranous tissues were stripped and homogenized in a sterile 10 mmol/L Tris buffer containing 1 mg/L aprotinin at 4°C. The homogenate was centrifuged, and the supernatant was frozen at -20°C; the precipitate was stored in 0.5% sodium lauryl sulfate, 0.5% β-mercaptoethanol, 1% glycerol, 2mmol/L phenylmethylsulfonyl fluoride, 10mmol/L Homogenize the N-hexylmaleimide in 50mmol/L Tris buffer, calculate the protein content, and store in aliquots at -80℃. Mice were immunized with Inner Ear Antigen (IEAg) prepared, and IEAg Complete Freund Adjuvant (CFA) emulsion was injected subcutaneously at the back of each model mouse, and pertussis was injected into the back of the foot to reduce the toxicity of pertussis Live bacteria 50μl (containing 25×1000000000 cells), IEAg content is 100μg. After immunization, the mice were tested for the auditory brainstem response (ABR) of bilateral ears: the animals were placed in an acoustic and electrical shielding room after anesthesia, and tested with an evoked potential meter. Take the stimulus 0.1ms as a short sound, the interval time is 50ms, the filter is 100～3000Hz, the scanning time is 10ms, and the superimposition is 500 times. White noise lower than 30dB of the stimulus will mask the side ear. The response threshold of wave II was used as the standard to judge the response threshold of ABR. Anti-IEAg antibodies in animal serum were detected at 7, 14, 21, 28 days after modeling, and temporal bone tissue was taken to make conventional sections of inner ear tissue. After HE staining, observe under light microscope.

　　(2) Model characteristics The body weight of the model animals decreased progressively within 2 weeks after immunization with IEAg. After 3 weeks of immunization, the body weight can return to the pre-immune level and increase slowly; the animals may still experience reduced activity, insensitivity to auricle reflexes, and walking Instability and other clinical manifestations. Before modeling, the average ABR response threshold of C57BL/6 mice was 46dB SPL, and there was no significant difference between the left and right ears; the animal ABR response threshold was significantly increased on the 7th day after modeling. The histopathological observation of the model animal microscope showed that there were more inflammatory cell infiltrations in the scala tympani, cochlear duct, vestibular scala and the blood vessels around the cochlear axis in the inner ear tissue, and more endolymphatic hydrops were seen in the first 2 weeks after modeling; and spirals were visible. The ganglion cells were swollen and necrotic-like changes, and the number of cells decreased progressively after immunization. After this model was made with guinea pig IEAg, the induced cell infiltration was limited to the inner ear tissue, and the hearing changes of model animals were significantly positively correlated with the degree of cell infiltration and serum anti-IEAg antibody levels, suggesting that immune and humoral responses are inducing autoimmune inner ear It may play an important role in autoimmune inner ear disease (AIED).

　　(3) Comparative medicine. The pathogenesis of autoimmune inner ear disease is still unclear clinically, and there are no specific laboratory test indicators. The treatment principle is controversial and the treatment effect is not ideal. Establishing a disease animal model similar to clinical AIED will help clarify the pathogenesis of autoimmune inner ear disease and analyze the effect of immunomodulatory treatment of inner ear. Previous studies reported that most experimental AIEDs used outbred guinea pigs as experimental animals, and used multiple booster immunizations after the initial systemic immunization, which was induced by the body's autoantibodies through humoral immune-mediated immune responses. The success rate of these models is low (20%-50%); the degree of hearing impairment is light (8-25dB); the pathological changes of the inner ear are not typical; the experimental period of multiple boosters is longer, which increases the uncertainty of the experiment. The impact of results. Because outbred animals are worse than inbred animals in terms of repeatability and detailed research of immunological analysis, these models cannot accurately reflect the pathogenesis and pathophysiological changes of human AIED. This model uses inner ear antigens to immunize animals with significant hearing loss, and the degree of hearing damage increases with the immune process. Autoantibodies against inner ear tissue can be detected in the serum. These changes are consistent with the clinical manifestations of AIED patients; morphologically Characteristic autoimmune damage such as perivascular inflammation can be further confirmed. As a widely used inbred mouse, C57BL/6 mice are very consistent among individuals and have the same response to experiments, which can eliminate the influence of heterozygous genetic background on experimental results. C57BL/6 mice can show high-frequency-related senile hearing loss with age, so they are used to study presbycusis. The AIED model induced by this method has the advantages of high morbidity, severe hearing loss, typical pathological changes of the inner ear, short modeling cycle, simple production method, relatively simple experimental conditions, and easy control of other factors.