| INTRODUCTIONHigh amounts of nitric oxide ( NO) generated by inducible nitric oxide synthase (iNOS) cause severe cellular injury and are relevant in the pathogenesis of inner ear disease. It has been demonstrated that NO is involved in the ototoxicity of noise, gentamycin, ischemia and inflammation. Inhabiting the expression of iNOS can attenuated inner ear damage caused by many reasons. Recent evidence suggests that sodium salicylate can reduce the expression of iNOS in cultured rat hepatocytes, cardiac fibroblasts and vascular smooth muscle cells. We presume sodium salicylate may reduce the inner ear damage by inhabiting the expression of iNOS. In this study we examined the effect of sodium salicylate on the expression of iNOS in rat cochlear treated with lipopolysaccharide immunohistochemically. The protective effect of sodium salicylate in inner ear was also assessed.MATERIALS AND METHODSMaterials: 24 healthy, otomicroscopically normal male Wista rats weighing 250 - 300g and with a normal Preyer reflex were used. During instillation and ABR measurement, the animals were anesthetized with 40mg/kg 2% sodium pentobarbital i. p. All instillations into the middle ear cavity were performed in the posterior - superior quadrantof the tympanic membrane under a Zeiss operating microscope. The material was divided into three groups of equal size. All the animals were exposed to LPS in the right ear and sterile saline in the left ear. The animals in two groups were given various dosage of sodium salicy-late 4hours before the injection of LPS. Methods; (1) ABR procedure. ABR measurements were recorded in all animals prior to inoculation, 12h and 24h afterwards. The measurement was performed in an acoustically shielded unit. The determination of ABR thresholds was through three subcutaneously inserted needle electrodes. The sound stimuli was filtered clicks with 10ms duration, rise and fall times of 1ms presented at a rate of 20/s, frequency range 100 -3000 Hz. Attenuation covered a range of 0 - 105 dB. A total of 128 stimuli were delivered and the responses fed into an averager and recorded on an X/Y plotter. During ABR recording, the body temperature was kept constant at 37 ± 0. 2℃. The threshold was determined by waves II 1H. (2) Immunohistochemistry. 4 animals from each group were sacrificed each time after ABR 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 animals 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. Decalcifica-tion 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 μm thickness with a microtome and were mounted on L - polylysine - coated sllides. After immersed in 3% H2O2 and normal goat serum, the sections were incu-bated with antibody to iNOS at 371! 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. Analysis and photography of the sections under Meta - Morph and Olympus light - microscope followed.RESOULTSABR thresholdsThe instillation of LPS obviously impaired ABR thresholds and the most severe alterations were observed in the group without application of sodium salicylate. The application of sodium salicylate significantly reduced the ABR threshold impairment caused by LPS. The ABR threshold shifts between groups with different dosage of sodium salicylate showed no difference. The instilla... |
PDF Full Text Request