| IntroductionThe ototoxicity of gentamycin can cause both cochlea and vestibular damage, and the damage of vestibular is more severe. How gentamycin takes effect is still unknown, and there are some reports prove that apoptosis plays an important role in the procedure, and nucleus shrinkage, chromosome condensation and apoptosis body formation such the marked character of apoptosis; Meanwhile, JNK (c-Jun N-terminal kinase ), a member of MAPKs (Mitogen-Activated Protein Kinase) family is found participate the apoptosis procedure. Our research try to figure out the character of gentamycin induced vestibular hair cell damage and its mechanism.Material and Method1. 30 healthy, otomicroscopically normal guinea pigs weighing 250-300g and with a normal Preyer reflex were used. Animals were divided into 2 groups with 15 in each group. Animals in experimental and control group received Gentamycin(100mg k g-1 d-1) and saline respectively in im. After successive 7 days, all animals were sacrificed.2. Hair cell preparation: all the animals were sacrificed, and the left temporal bones of each animals were removed. Round window and oval window were perfumed with AgN03 and fixed with 4% paraformaldehyde. After exposed under sunlight 2-3 hour, the cristea ampullae was taken out and observed under microscope.3. Slides preparation: All animals from each group were sacrificed under deep anesthesia and fixed via cardiac perfusion with 4% paraf ormaldehyde after flushing out the red blood cells with 0. 1 m PBS. Right temporal bones of each animals were removed. The tissues were immersed in the same fixativesolution for 24h. Decalcification of the bone 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 12h. The specimens were reduced to sectional series of 10 um thickness with a microtome and were mounted on APES. Half of the specimens were detected by TUNEL system, and the other were analyzed by histochemistry.4. Immersed the slides in 0.2% Triton X-100 solution for 5 minutes, then rinse slidesby fresh PBS 5 minutes and again. Cover the slides with Equlibrat ion Buffer for 5 minutes and then add 50 ul of TdT for 60 minutes. At last, stained sample by DAPI.5. After immersed in 3% H2O2 and normal goat serum, the sections were incubated with antibody to P-JNK at 37C 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.ResultHair cell preparation: The HCs of experimental group shows swollen, loss its normal structure. The HCs of control group have clear margin, and arrange uniformly.Fluorometric result: The cells stained with green light which marked TUNEL positive is found in the experimental group, and locate at the cell layer adjacent to the epithelium; there is no such cells found in the control group.Immunohistochemical stain result: the HCs of experimental group shows dark yellow particle immune expression which mainlylocate at the apex of cristae ampullae; but the basal HCs which arranged uniformly show no such reaction, neither do the ones of control group.Comparision of fluorometric and histochemical result: the histochemical positive-stained cells can be seen in the fluorometric stained positively group, and the positive-stained cells locate at at the same place: the apex of cristea ampullae, we can say these cells are vestibular hair cells from their distributional character; slides, which come from fluorometric negative-stained group, show no histochemical positive stained cell.CONCLUSION(1) Systemic use of gentamycin can damage vestibular...
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