| INTRODUCTIONThe JNKs are a member of the mitogen activated group of protein kinases that are important signal transduct enzymes involved in many facts of cellular regulation including apoptosis. The ototoxic potential of aminoglycoside antibiotics is well known, an increasing body of evidence suggests that HC death after aminoglycoside ototoxicity can occur through apoptosis. Amikacin, a new type of semi - synthetic wide spectrum antibiotics, gains satisfying result in clinical use, but the ratio of hearing loss is 15. 7%. Our research try to figure out the ototoxic character of amikacin and the expression of p - jnk in hair cells ( HCs) , discussing the ototoxic mechanism through DPOAE, cochlea preparation and im-munohistochemistric method.MATERIALS AND METHODSMaterials: 12healthy, otomicroscopically normal guinea pigs weighing 250 - 300g and with a normal Preyer reflex were used. Animals were divided into 2 groups with 6 in each group. Animals in experimental and control group received amikacin (300mg kg-1 d-1) and saline respectively in im. After successive 7days ,all animals were sacrificed. All instillations into the middle ear cavity were performed in the posterior - superior quadrant of the tympanic membrane under a Ziss operating microscope. Methods: (1) Assessment with DPOAE: In sound - proof room, we assess the HC function of guinea pigs under deep anesthesia with DPOAE (Intelligent Hearing Systems, USA). The two applied stimulus pure tones is characterized by its frequency and sound intensty f2/f1 =1.22 , L1 - L2 = 10dBSPL, and we apply SNR beyond 3dB as detectory standard. Wecollect the data of amplitude when frequenry f0[f0 = (fj x f2)1/2] is 1N2N4N6N 8kHz. (2) Immunohistochemistry. All animals from each group were sacrificed after the 2nd DPOAE measurement and fixed via cardiac perfusion with 4% paraformaldehyde after flushing out the red blood cells with 0. 1 m PBS. Both temporal bones of each animal were removed. The bone near the apex, the round and oval window - membrane were opened for better penetration of the fixative. The tissues were immersed in the same fixative solution for 24 h. Decalci-fication of the cochlear was performed in 10% formate - sodium formate for a week. Before embedded in OCT for immunohistochemical analysis, the tissues were dehydrated in 30% saccharose for 24h. The specimens were reduced to sectional series of 10 jxm thickness with a microtome and were mounted on APES. After immersed in 3% H202 and normal goat serum, the sections were incubated with antibody to P - JNK at 37 ^C overnight. A biotinylated anti - rabbit antibody was used as the second AB. For negative controls the primary AB was omitted on one of the mounted sections on all slides. Processing was ultimately performed with a SABC and DAB. The reaction was observed under a light - microscope and was finally stopped by application of PBS. The specimens were dehydrated in baths of ascending ethanol concentration. The primary antibody was replaced with PBS as a control for non - specific binding. ( 3) Cochlear preparation: all the animals were sacrificed after second DPOAE measurement, and the left temporal bones of each animals were removed. Cochlea were perfused with AgN03and fixed with 4% paraformaldehyde from the apex. After exposed under sunlight 2-3 hour, the basal membrane was taken out and observed under microscope. The experimental data were analyzed by SPSS using t test.RESOULTSAssessment with DPOAE: the DPOAE amplitude of experimental animals decreased refer to pre - injection except 1kHz; the decrease amplitude on 2, 4, 6, 8kHz has a significant difference between the experimental group and the control one.Cochlea preparation: The individual inner HCs of experimental group swollen , the cilia of outer HCs miss partially, and there are spaces between the outer HCs which should have arranged uniformly. The damage of outer HCs mainly located in the basal turn and the second turn. The outer hair cell of control group shows no anomaly: had a clear structure and arranged uniformly.
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