Expression Of Caspase-3 On The Cochlear Hair Cells' Apoptosis Of Guinea Pig With Gentamycin Ototoxicity

INTRODUCTIONAminoglycoside antibiotics are in common use wide - compose antiseptic and are used to treat tuberculosis and G- bacterium infection disease. But its toxicity to cochlear, vestibule and kidney is obvious, which restricts its applications in the clinic. Lately, some researches indicate aminoglycoside antibiotics can induce hair cells ( HCs) quick apoptosis on the cochlear, apoptosis have something with Caspases family activation. Through inspecting expression of Caspase - 3 on the cochlear hair cells' apoptosis of guinea pig with gentimycin ototoxicity in this experiment, to discuss cochlea hair cells'damnification characteristic and ototoxic mechanism.MATERIALS AND METHODSMaterials: 30 healthy, otomicroscopically normal guinea pigs weighing 250 ~ 300g and with a normal preyer reflex were used. They were divided into 3 groups with equality. Experimental No. 1 group: gentimycin 200mg kg-1 d-1; Experimental No. 2 group: gentimycin 200mg kg-1 d-1 plus to fusaimi 80 mg kg-1 d-1; Control group: 0.9% natriichloridi 200mg kg-1 d-1; Respectively abdomen injection, after successive 6days, all animals were sacrificed on the seventh day.Methods: (1) Assessment with DPOAE; In sound - proof room, we assess the HC function of guinea pigs under deep anesthesia with DPOAE . The two ap-plied stimulus pure tones is characterized by its frequency and sound intensty f2/ f1 = 1. 22, L1 - L2 = 10dBSPL, and we apply SNR beyong 3dB as detectory standard. Wecollect the data of amplitude when frequency f0 = (f1× f2)1/2 is 1, 2,4,6,8kHz. (2) Cochlear preparation: 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. 1m 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 24h. The specimens were reduced to sectional series of 10 μm thickness with a microtome and were mounted on APES. (3) Immunohistochemistry. After immersed in 3% H2O2 and normal goat serum, the sections were incubated with antibody to Caspase - 3 at 37℃ overnight. A biotinylated anti - rab - bit antibody was used as the second AB. For negative control 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. (4) Inspecting apoptosis; According to Wu Han Boster Company providing TUNEL reagent box explanation. The experimental data were analyzed by SPSS 12.11 using test.RESULTS1. Assessment with DPOAE: the DPOAE amplitude of experimental animals decreased refer to pre - injection expect 1kHz. Between the experimental group and the control group, the experimental NO. 1 group and the NO. 2 group, the DPOAE amplitude decreased refer to pre -injection expect lkHz, having a significant difference.2. Cochlear preparation; Many HCs of experimental group swollen, the cilia of outer HCs miss partially, and there are spaces between the outer HCs which should have not arranged uniformly. The damage of outer HCs mainly located in...