动物模型,中耳炎 z-bo 2020-08-08 663

　　1 Guinea pig secretory otitis media (Secretory Otitis Media, SOM) model

　　(1) Copy method Use guinea pigs weighing 300～420g and normal Preyer reflex to perform otoscope examination first to exclude external and middle ear diseases, and test 0.5, 1, 2, 4kHz 100dB short pure tone ear reflex respectively threshold. Inject pentobarbital sodium into the abdominal cavity at a dose of 30 mg/kg body weight for anesthesia. After anesthesia, lie on the side and fix the guinea pig’s right external auditory canal. The external auditory canal is disinfected with 2% iodine tincture and 75% ethanol to inactivate the pneumonia with 3×108 CFU/ml 0.1ml of streptococcal suspension is injected into the tympanum of the animal through the front and lower parts of the tympanic membrane, and cultured on agar medium before injection to ensure that no live strains grow to prevent acute infection. Model animals undergo otoscope examination every day after injecting the bacterial suspension, observe for 2-10 days, pay attention to the color changes of the tympanic membrane, and recheck the auricular reflex threshold. Subsequently, the animals were sacrificed after anesthesia, and the bilateral auditory vesicles were taken, fixed with formaldehyde and de-sheathed, and the sections were embedded in paraffin, stained with conventional HE, and observed under a light microscope.

　　(2) Model characteristics 48 hours after the injection of the bacterial suspension, the tympanic membrane of the model animal has different degrees of radial expansion of the vascular pattern. On the 3rd day, the tympanic membrane loses its normal luster and is pale yellow, brownish red or amber. The most significant performance was on the 7th day, the tympanic membrane activity was reduced, and the middle ear cavity had a light yellow transparent thin exudate, mainly serous, thin and watery. The pure tone response of model animals changed to different degrees, mainly 0.5, 1, 2kHz. Histopathological observations under the microscope showed that inflammatory cell infiltration, dominated by lymphocytes, could be seen in the middle ear cavity of animals, with lymphocytes, monocytes, phagocytes, and eosinophils in some cases; auditory vesicle wall mucosal congestion and edema, The capillaries increase, the permeability increases, and the mucosal epithelial cilia fall off.

　　(3) Comparing medical secretory otitis media is a common clinical disease in upper otology, and it is also known as an "intractable disease in otology". It has a high incidence and severe hearing loss, which often leads to middle ear adhesions, tympanic sclerosis, and Cholesterol granuloma, etc., cholesteatoma may even occur in individual cases. For the research on the etiology and pathogenesis of SOM, no convincing conclusions have been drawn so far, and there is no exact and effective prevention method. In recent years, with the development of molecular biotechnology, the detection level and the continuous improvement of culture methods, many research reports have successively detected pathogenic bacteria from the middle ear effusion, confirming the presence of bacterial DNA in the middle ear effusion. Therefore, infection and inflammation of the middle ear cavity are the main causes of SOM, and this view is gradually being accepted by people. The results of bacteriological and histological examinations and clinical signs have shown that SOM may be a mild or low-toxic bacterial infection of the middle ear, and the bacterial decomposition product, endotoxin, plays a certain role in the pathogenesis of SOM. . This model uses an inactivated Streptococcus pneumoniae suspension to prepare a guinea pig SOM animal model. The operation is simple and the method is reliable. The results prove the production of SOM. The attenuated and inactivated Streptococcus pneumoniae used for modeling did not cause acute middle ear Inflammation is an ideal animal model for studying the pathological changes of SOM on hearing threshold and middle ear mucosa.

　　2 Mouse secretory otitis media model

　　(1) Copy method Select BALB/c mice with sensitive ear reflex, weight 25-30g, and 6-8 weeks old, and it is confirmed by otoscope examination that there is no external ear canal and middle ear infection. BALB/c mice were given 1.2 mg ovalbumin (ovalbumin, OVA) dissolved in 0.6 ml phosphate buffered saline (PBS), and 5.14 mg aluminum hydroxide was used as immune adjuvant for intraperitoneal injection, once a week, for 2 consecutive weeks , Systemic sensitization; 2 weeks later, 20% urethane anesthesia was injected intraperitoneally at a dose of 5ml/kg body weight. Aseptically, 15μl of 1g/L OVA+PBS mixture was injected into the tympanic cavity through the anterior and lower tympanic membrane. Anesthetize, observe the state of the tympanic membrane, and repeat the injection 2 times to stimulate in the ear; creating a model of secretory otitis media. Model animals undergo otoscope examination every day after intra-ear injection of OVA solution. Two days after the second challenge, the animals were sacrificed, the bilateral auditory vesicles were taken, routinely fixed and decalcified, and pathological tissue sections were made. The hyperplasia of tympanic mucosa was observed under HE staining light microscope, and the thickness of middle ear mucosa was measured, and eosinophils were counted. Cells, plasma cells, mast cells were counted by toluidine blue staining.

　　(2) Model characteristics Model animal otoscope examination results show that 24 hours after injection, the tympanic membrane and surrounding tissues appear erythema, 36 hours later, the tympanic membrane loses its normal luster and is pale yellow or amber, and the tympanic membrane activity is restricted by tympanic otoscope examination. But he could not see the level of clear liquid through the tympanic membrane. Most model animals can find a light yellow transparent thin liquid in the middle ear cavity. In the middle ear of model animals, a thin, watery serous exudate was seen, which was light yellow, the tympanic mucosa was congested and edema, capillaries increased, permeability increased, and epithelial cilia fell off; microscopic histopathological observation showed that the submucosa propria In the bone marrow cavity, inflammatory cell infiltration dominated by infiltration of eosinophils, plasma cells and lymphocytes appears. Mast cells and monocytes are still visible in the inflammatory cells. Toluidine blue staining on the auditory vesicle wall of model animals showed blue nuclei and reddish-purple mast cells, which were mainly distributed around the capillaries in the submucosal connective tissue. The model is easy to make and the method is reliable.

　　(3) Comparative Medicine Secretory otitis media is a common disease in otology, which occurs mostly in children and has a high incidence. Its pathogenesis is still unknown, and there is no exact and effective method for prevention and treatment. In recent years, with the progress of immunology and molecular biology research, the theory of the body's middle ear as an independent immune defense system has gradually been accepted by most scholars; the current immunology viewpoint has entered the field of otology, and the body's ear is not an immune exemption site. The entire middle ear mucosa continues with the upper respiratory tract mucosa of the nasopharynx, and covers the eustachian tube, tympanum, tympanic sinus and mastoid air chamber. As a derivative of the upper respiratory tract, the mucosal system of the middle ear also has the function of the upper respiratory tract mucosa; Once a potential immune organ is stimulated by a pathological antigen, it can activate various immune cells in the mucosa to produce a local immune response. This model is a secretory otitis media model made by OVA sensitization and in-ear stimulation. The results confirmed that eosinophils and mast cells related to type I allergies were involved, and indirectly confirmed that type I allergies are involved in SOM. Role in pathogenesis. The SOM animal model established by this method provides a good experimental model for further in-depth study of the correlation between SOM and type I allergy.

　　3 Rat Suppurative Otitis Media (Suppurative Otitis Media) model

　　(1) Copy method Male SD rats weighing 220-320g have normal ear reflex, complete tympanic membrane, clear light cone, good light transmittance, and auditory brainstem evoked potential threshold of 20-30dB. Rats were anesthetized by intraperitoneal injection of sodium pentobarbital at a dose of 35 mg/kg body weight. Method ①: Under an operating microscope, sterilize the external auditory canal of the model rat with 75% ethanol, and inject 0.05ml of the pre-prepared pneumococcal suspension with a concentration of 5×1000000CFU/ml into the lower quadrant of the anterior tympanic membrane on the right side, and inject 0.05ml sterile into the left ear The suspension was used as a control. Method ②: The rats were cut through the ventral side of the head and neck of the rat, and the skin was pulled to both sides to separate the buccal muscles and the posterior part of the mandibular digastric mandibular muscles, along the myofascial space, behind the buccal muscles. There are thin and brittle blisters on the midline side of the neck on the 3 deep sides, which are the auditory vesicles, which can be positioned by pulling the ears. Use a small puncture needle to inject the suspension on the left and right sides in the same way as ①. After the operation, the rats were bred routinely, and they were free to drink and eat. At 7d and 14d postoperatively, after observing the tympanic membrane of the model animals, they were perfused with formaldehyde-ethanol intracardiac for 10 minutes, and then the head was placed in the formaldehyde-ethanol fixative for 1 week. Take bilateral auditory vesicle tissue specimens, make conventional tissue sections and SEM specimens, and observe under light microscope and electron microscope.

　　(2) Model characteristics At 7d and 14d postoperatively, the tympanic membrane of the model animal was obviously thickened and showed yellow purulent changes. Observation under anatomical microscope showed that the animal's auditory vesicles were filled with yellow objects, and histopathological observation under microscope It showed that the mucosal layer was obviously thickened, and there was no adhesion formation; scanning electron microscope observation showed that the mucosal surface layer was obviously thickened, and there was no adhesion formation. The model making method is simple and easy to implement, and the result is reliable.

　　(3) Comparative Medicine Rats are very similar to the tympanic cavity of the human middle ear in terms of auditory bubble anatomy and mucosal structure. They are good experimental animals for research on middle ear diseases. However, the rat ear canal is located in the lower part of the external auditory meatus, close to the ventral side, and its back has many muscle structures. It is difficult to find the way from the dorsal side. It is different from the guinea pig ear through the back of the head, which is easy to find. The model made by this method adopts a midline approach through the ventral side of the head and neck of the rat, and the auditory vesicles are easily found in the myofascial space between the buccal muscles on both sides and the posterior part of the mandibular digastric muscle. In view of the fact that the bone quality of the rat auditory vesicle can affect the puncture, separation and sectioning, the rats with a body weight of about 250g and thin bone of the auditory vesicle should be selected when making this model. In recent years, through clinical pathological biopsy, serial section observation of temporal bone specimens, and animal experiments, it is generally agreed that various types of otitis media are manifestations of the same continuous inflammation and repair reaction process at different stages, and various types of otitis media can coexist. Clinically, adhesive otitis media is chronic otitis media or its sequelae and complications. It has a high clinical incidence and is an important cause of deafness in patients. It has a major impact on human health and quality of life, especially in children. Affect growth and development. However, because the pathogenesis of adhesive otitis media is still unknown, and the clinical efficacy is not good, it is still a major clinical problem in otology. Therefore, the establishment of an ideal adhesive otitis media animal model is of great significance for understanding its pathogenesis and providing effective treatment methods. The establishment of this model provides animal experimental evidence for further replication of adhesive otitis media animal models.