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Effect of Sodium Salicylate on the Expression of Inducible Nitric Oxide Synthase in Rat Cochlear Treated with Lipopolysaccharide

Author: MuEn
Tutor: JiangXueJun
School: China Medical University
Course: Otorhinolaryngology
Keywords: Sodium salicylate Bacterial endotoxin Cochlea Inducible nitric oxide synthase
CLC: R764
Type: Master's thesis
Year: 2003
Downloads: 34
Quote: 0
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Abstract


Introduction of inducible nitric oxide synthase (inducible nitric oxide synthase, iNOS) the catalytic synthesis excessive nitric oxide (nitric oxide, NO) can cause extensive tissue damage, and to participate in the noise, ototoxic drugs, ischemia, inflammation causes cochlear pathological processes. The inhibition of iNOS can reduce the damage of the inner ear of a variety of causes. The study found that sodium salicylate can inhibit liver cells, cardiac fibroblasts in vitro, iNOS expression in vascular smooth muscle cells, suggesting that sodium salicylate may play a protective effect in the inner ear through the inhibition of iNOS. In this study, using immunohistochemistry to observe the effects of sodium salicylate expression of the rat cochlea iNOS caused by bacterial endotoxin (lipoplysaccharide, LPS), and to investigate the protective effect of sodium salicylate on cochlear. Materials and Methods Materials: Take auricle reflex normal, weighing 250-300g healthy male Wista rats 24 ear the microscopic examination eardrum exclude otitis media. All animals were divided into 3 groups, respectively LPS group, NaSA / LPS group and NaSA / LPS group. Eardrum to The right tympanic injection concentration of 5mg/ml the LPS0.5mg, the left tympanic injection of normal saline. NaSA / LPS group and NaSA / LPS animals 4 hours prior to injection of LPS by intraperitoneal injection of salicylic acid sodium 200mg/kg and 400mg/kg respectively. LPS group animals intraperitoneally injected with saline. Methods: (1) ABR detection: before the injection of LPS, 12 and 24 hours after injection test ABR thresholds. Alternative animal in 2% sodium pentobarbital 40mg/kg anesthetized by intraperitoneal injection, in soundproof electrical shielding indoor conventional methods to test ABRs recorded the reaction threshold. Line a short sound (click) Danac-7 type sound stimulus is stimulus, frequency 20 times / sec, scan time 10ms, filtering 100-3000Hz, Acoustic Output range 0-100dB, signal superimposed 128 times, to Ⅱ Ⅲ wave response threshold for the standard judge hearing threshold. *) Immunohistochemical examination: after LPS injection after each test ABR in each group of four animals were sacrificed. Thoracotomy under anesthesia at 4 ° C saline via cardiac perfusion, out of all the red blood cells more than 4% paraformaldehyde perfusion fixed. Decapitated, quickly remove the bulla, isolated cochlear snail sharp punch, open round window. Oval window, the cochlea in the same fixative 24 / J \\ time. Formic acid-sodium formate solution decalcification week, replace the decalcification solution daily. And then transferred into a 30% sucrose solution at 4 ℃ overnight. OCT-embedded along the worm shaft direction parallel line 10pM thick slices ready for immunohistochemical staining the visions ABC legal person to be sliced ??and dried in the air, 3% hydrogen peroxide solution for 10 minutes to remove endogenous peroxidase; normal goat serum at room temperature 20 minutes closed; antibody (rabbit anti-mouse iNOS monoclonal antibody overnight at 4 ℃; combined into incubated at room temperature for 20 minutes plus streptavidin and biotin (biotinylated goat anti-rabbit IgG secondary antibody a peroxidase complex (611-PP the coffee PM10n-M. roiloi4PPC MPL, the SABC was 7t reaction for 20 minutes; diaminobenzidine chromogenic light microscope, controlling the reaction time, distilled water to terminate the color, dehydrated in a graded alcohol xylene, mounted with neutral gum light microscope. blank control antibody was replaced with PBS, the remaining steps unchanged. using the Meta Morph electronic computer image analysis system, the determination of the average gray value of the of iNOS positive reaction was to mean on the standard deviation, the gamma value, the lower the degree of group reaction. the ABR thresholds mean standard deviation of the results of analysis of variance using the SAS statistical analysis software, significant difference Results * injection of the right ear of Li ABR thresholds was significantly higher, of which the most significant L.e group, NaSA / off an elevated hearing threshold of the two groups was significantly lower than the Li group by one-way ANOVA parallel q test found the LPS group NaSA / LPS, two groups ABR threshold shift significantly compared to the differences with significant O <0.05 * Shui A / fat a ** R threshold shift between the two groups had no significant difference. left ear injection of saline ABR threshold slightly elevated, light significant difference between groups. 2. injection e observed under an optical microscope, vascular · 2 · pattern in the cochlear spiral ligament, spiral ganglion Ke iNOS expression, etc. the supporting cells of alternate's strongest, NdA / fat group followed of N aSA / aSA / classes two weakest, only saline injection contralateral cochlear various parts without expression in LPS group iNOS expression image analysis: NASA / m, the average gray value of the positive reaction of the two groups of cochlear iNOS significantly higher than the registered group, significant differences in the statistical analysis of (one-way ANOVA, P <0.05, NaSA / m, two groups of cochlear iNOS activity was significantly lower than LPS group. discussion the intratympanic bacterial LPS-induced experimental animal hearing loss with cochlear iNOS expression increased, suggesting that NO may be involved in the inflammatory process induced cochlear injury; pre-injection NaSA LPS-induced ABR threshold shift the degree has been reduced, while the expression of iN-OS also weakened proved NaSA LPS induced cochlear injury has a protective effect, this protective effect may be related to the inhibition of iNOS expression has been known vivo three isoforms of NOS, namely the neuronal hNOSX endothelial * NOS) and inducible ONOSX which two types of structure type * NOS physiological conditions that exist, catalyzed by a small amount of NO synthesis, involved in neurotransmission, blood flow regulation, and the immune response, and other physiological activities of iNOS under normal circumstances, not express expression started when the stimulation by LPS, tumor necrosis factor and other factors, and its activity is thousands of times more than the other two subtypes, can catalyze the synthesis of large amount of NO produce toxic effects on cells and tissues. Studies have shown that excessive NO synthesis in the cochlea of ??iNOS expression and its catalytic the cochlear auditory function can produce inhibitory. cochlea by LPS, tumor necrosis factor-stimulated visible expression of iNOS ototoxicity drugs such as gentamicin * platinum can also be caused by cochlear iNOS expression in animal models of noise-induced hearing loss can also be seen in iNOS expression in the cochlea, the application of NOS inhibitors such as N-nitro-L-arginine acid teams NNA J nitro-arginine methyl ester can reduce the cochlea iNOS expression and NO synthesis under the above conditions to participate in the ear hearing dysfunction caused by hypoxia or ototoxic drugs

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