Olfactory Ensheathing Cells Promote Injury Of Rat Spiral Ganglion Cell Protection And Repair Of Experimental Research

Part 1 Culture, purification, label and identification of olfactory ensheathing cellsObjective:To culture, purify and label OECs. Methods:The capsule of new born rat olfactory bulb was dissected. The tissues were minced with a razor blade, and using trypsin. OECs were obtained and purified based on their special rate of attachment which was different from the other harvested cell types during culture. DF12 medium supplemented with NGF, b-FGF and 10% FBS and antibiotics were employed to culture the OECs. Half of the medium in each group was refreshed three times a week. OECs were immunocytochemically characterized and confirmed by expression of P75NTR and GFAP. OECs nucleus were labeled with Hoechst33342. Results:OECs from the olfactory bulb of new born SD rats were highly purified based on the differing rates of attachment of the various cell types. Cultured OECs displayed a typical spindle-shaped soma with long and slim processes.92 percent of the cells cultured were P75NTR and GFAP immunoreactive. About 100 percent cells were labeled with Hoechst 33342. Conclusion:The high pure of olfactory ensheathing cells can be harvested using special rate of attachment which was different from the other harvested cell types during culture and facilitate the research on the olfactory ensheathing cells in vitro.PartⅡTo establish a Model of ototoxicity of kanamycin sulfate in adult ratsObjective:to assess the ototoxicity of KM in adult rats. Methods:Sixty male rats (6-7 weeks old) were randomly divided into three groups: the experimental group one, the experimental group two and the control group. The animals in the experimental group one were injected intraperitoneally with KM (100mg/ml) in 500 mg/kg per day consecutively for two weeks, and KM (80mg/ml) in 400 mg/kg per day in the experimental group two, while in the control group equal volume of normal saline was used in the same way. The ABR was recorded to monitor the changes in hearing thresholds. To assess the ototoxic condition of the cochlea, we examined the density and morphology of HCs and SGCs using surface preparations and frozen sections. Results:The pathological change was similar in the two experimental groups, just that the change in the experimental one was more severe than that in the experimental two. The hearing threshold of rats in the experimental group one was elevated by more than 60 dB across all the frequencies two weeks after administration of KM, while that was elevated by more than 20dB in experimental group two, especially in 32000Hz, the ABR threshold was elevated by more than 30dB. And the density of SGCs became lower, with the more severe loss of OHCs than IHCs. Conclusion:The 6-7 weeks old rats that had been injected with KM in the dose of 400 mg/kg per day for two weeks were suitable for the transplant of OECs.PartⅢResearch on olfactory ensheathing cells protecting damaged spiral ganglion cellsObjective:to observe the protective and prothetic effect of the transplanted OECs on the impaired SGCs in rats. Methods:the OECs which had been cultured for 7 days were labeled, and then they were transplanted into the cochlea of the experimental group rats in the concentration of 1×106/ml, while in the control group equal volume of D-Hanks was injected into cochlea. And all these rats had been injected intraperitoneally with KM 400mg/Kg per day consecutively for 2 weeks beforehand. The ABR was recorded to monitor the changes in hearing thresholds. To assess the ototoxic condition of the cochlea, we examined the density and morphology of SGCs using frozen sections. Results:there was no obvious difference between the ABR thresholds of the rats in the control and the experimental group at each frenquency. There were a number of blue cell nuclears labeled with Hoechst 33342, distributing from the bottom to the top of the cochlea in the experimental group. The density of the SGCs of the rats in the experimental group was obviously different from that in the control group: most of the SGCs in the experimental group were in good shape, arranged in a closer manner, and the nuclear membrane, the chromatin and the nucleolus were all clear. Only a small number of the chromatin verged or become necrotic, but the density was much lower than that in the normal rats. The SGCs in the control group died in a great number, some material in the remnant nucleus was phagocytized by macrophages. Most cells ranked in a loose and disordered manner; only a few normal SGCs in this group could be seen. A lot of fragments of the necrotic SGCs were seen in the control group by means of immunofluorescence. Conclusions:OECs could increase the survival rate of SGCs, playing a protective role on the impaired SGCs in rats.