The Apoptosis On Cochlea Hair Cell Induced By The Oxygen Free Radical In Neonatal Rats

Excessive oxygen free radical(sOFR)may produce severe cell toxicity, they also can cause the instant or irreversible damage. The cell apoptosis is a process that involves many kinds of physiological and pathological factors, and many inflammation media and cell factors all affect the process. It is very important that the production of massive OFR and the late response induced by oxidative stress for the process of cell apoptosis. Many studies have indicated that the deafness resulting from drug toxicity,noise-induced hearing loss ,ischemical reperfusion deafness and presbycusis all have relationship with OFR. In these deseases ,ROS is all been invovled and it may cause apoptosis or necrosis of cochlea hair cell. So the mechanism of OFR-induced apoptosis on hair cell has very important significance. It has been suspected that OFR can induce apoptosis in many experiments in vivo, but the apoptosis model directly induced by OFR in vitro hasn't been seen. It is still not clear that the way caused the cochlea hair cell depletion. To seek for effective natural antioxidants and try to activate endogenous antioxidants will provide evidence for the treatmen of sensorineural hearing loss. In this experiment ,we set up a damage model of OFR by tissue culture methods of organ of Corti in neonatal S.D. rat. The apoptotic hair cells of cochlea were detected. N-acetylcysteine (NAC) works in the extracellular environment, and is a free radical scavenger and a precursor of intracellular cysteine and glutathione (GSH). NAC prevent apoptosis caused by many kinds of injury. In this study, the inhibit effects of NAC on OFR-induced apoptosis were studied in vitro. The study is divided into the following three parts. Part one: To set up tissue culture methods of organ of Corti in neonatal S.D. rat and investigate the application of AO/PI staining on the detection of cochlea hair cell. Twenty-four hours after the tissue cultivation, new epithelia cells and fibroblast were found around the tissue. Inner hair cells(IHCs) ,outer hair cell(OHCs) and supporting cell grew well. The tissue was stained by Acridine orange(AO) and Prodium iodine(PI). It becomes green,which means the tissue has good activity. The culture of organ of corti can be an ideal method for otology research. Part two: To observe the apoptosis on organ of Corti induced by OFR in vitro. Forty-eight hours after the tissues cultivation, the tissues was stained by AO and PI. We observed the ratio of apoptotic cells in difffent groups. There was a higher ratio of apoptotic cells on basal outer hair cells compared with apical hair cells and middle hair cells. In every group, OHCs had a higher ratio of apoptotic cells than IHCs. Our results suggest OFR can cause apoptosis of hair cells, and the way it induced cochlea damage is dose-dependent furthermore. The basal hair cells may be more vunerable to OFR damage than apical hair cells. OHC are more susceptible to OFR than IHC. Supporting cells have considerably more survival capacity than sensory cells. It is possiblely because of the Ca2+ overload and the decrease of anti-oxidized ability that H2O2 induced the apoptosis on hair cell. Part three: To investigate the inhibit effect of NAC on OFR-induced apoptosis of cochlea hair cells. We observed the ratio of apoptotic cells in difffent groups. The ratio of apoptotic cells was significantly decreased by addition of the radical scavenger NAC. The protection by antioxidants implies that the apoptosis of hair cells is duo to OFR damage.In conclusion, the culture of organ of corti can be an ideal method for otology research. OFR can induce apoptosis of hair cells. The morphological change is consistent with free-radical mechanisms involved in the ototoxic side effects of aminoglycosides and noise-induced hearing loss. The effectiveness of antioxidants in improving the viability of hair cells indeed supports free-radical mechanisms as the ditermining cause of cell apoptosis. The biochemical basis of this phenomenon, susceptibility to free radicals, may offer further insight into ot...