Changes Of Bcl-2, Bax And Fas Experssion In Rat Auditory Cortex And Effect Of Cattle Encephalon Glycoside And Ignotin Injection After Auditory Depriviation

Objective:To study the changes of Bcl-2, Bax and Fas in auditory cortex of SD rats after auditory deprivation and the effects of the cattle encephalon glycoside and ignotin injection through the establishment of auditory deprivation(AD) animal model via bilateral cochlear damage technique. The research on expression change and plasticity of the auditory center after AD is to provide animal experiment information for the adjuvant treatment of patients with hearing impairment.Methods: 1 We chose 64 SD male rats that were 3 weeks old, healthy, no external auditory canal or middle ear infection, and had sensitive pinna reflex. They were raised in a clean and good environment with suitable temperature. They were randomly divided into 8 groups according to weight(8 rats per group): 7-day control group; 7-day operation group; 14-day control group; 14-day operation group; 14-day treatment group; 28-day control group; 28-day operation group and 28-day treatment group. Rats in operation groups were performed bilateral cochlear damage operation; rats in control groups were given only incision behind the ears; and rats in treatment groups were performed bilateral cochlear damage operation and given intraperitoneal injection of cattle encephalon glycoside and ignotin injection(1 ml/kg) daily for 14 days or 28 days after bilateral cochlear damage. 2 The rats in all groups were given auditory brainstem response(ABR) test 7 days after surgery. After raising the rats to the corresponding time points under the same condition, we randomly selected 4 rats from each group to have their bilateral auditory cortex for HE staining and immunohistochemistry, and to have the auditory cortex of the remaining 4 rats in each group forreal-time fluorescence quantitative PCR and Western blotting. 3 The morphological changes in rat auditory cortex were observed by use of HE staining method. 4 The expression of Bcl-2, Bax and Fas was measured by immunohistochemical method, and the positive rate of brown granular cells were counted in the microscopic view. 5 The expression of Bcl-2, Bax and Fas m RNA was detected using RT-PCR technique. 6 The expression of Bcl-2, Bax and Fas protein in rat auditory cortex was detected with Western Blot technique.Results: 1 ABR test : SD rats in the control groups had repetitive waves at 40 SPL d B, and rats had no repetitive waves at 90 SPL d B 7 days after cochlear damage operation, which proved animal model of auditory deprivation were successful. 2 General status after operation: The next day after auditory deprivation rats could drink and eat, crawl along a straight line, and maintain the balance when carrying the tail straight up. However, their movement was still sluggish, insensitive to external sound stimulation, and lack of pinna reflex compared with the rats in control groups which had sensitive pinna reflex. 3 HE staining results : There were varying amounts of apoptotic or dead cells, shrinked, dark dyed, pale colored cytoplasm, nuclear pyknosis, condensated and broken chromatin, and disappeared nucleoli in the operation and treatment groups. There were less apoptotic or dead cells, and lighter red-staining cytoplasm, reduced nucleus and chromatin aggregation in the treatment groups compared to the operation groups. Experimental results show that apoptosis phenomenon appear in rat auditory cortex after AD, and cattle encephalon glycoside and ignotin injection can reduce the degree of apoptosis. 4 Immunohistochemical results: There were Bcl-2, Bax and Fas-positive cell in all groups. The number of Bcl-2 positive cells in operation groups were much lower than that of the corresponding control groups, the number ofpositive cells were significantly higher in the treatment groups than that of the corresponding operation groups. Howerver the number of Bax and Fas positive cells were significantly higher than that of the corresponding control groups; and the number of positive cells were significantly lower in treatment groups than that of the corresponding operation groups. Experimental results show that the expression of Bax and Fas in rat auditory cortex after AD increased, the expression of Bcl-2 decreased, and cattle encephalon Glycoside and ignotin injection can prevent these changes. 5 Western Blot test results: Bcl-2 protein gray ratios in operation groups were significantly lower than those of the control groups; gray ratios in treatment groups were significantly higher than those of the operation groups. Howerer Bax and Fas protein gray ratios in operation groups were significantly higher than those of the control groups; gray ratios in treatment groups were significantly lower than those of the operation groups. The results show that Bax and Fas expression in rat auditory cortex after AD increased, Bcl-2 protein expression decreased, and cattle encephalon Glycoside and ignotin injection can reduce the extent of these changes. 6 RT-PCR results: Bcl-2 m RNA expression in operation groups was significantly lower than that of the corresponding control group; while Bcl-2 m RNA expression in treatment group was significantly higher than that of the corresponding operation group. However Bax and Fas m RNA expression in operation groups was significantly higher than that of the control group; Bax and Fas m RNA expression in treatment group was significantly lower than that of the corresponding operation group. Experimental results show that Bax and Fas(pro-apoptotic gene) m RNA expression in rat auditory cortex after AD enhanced, expression of Bcl-2 m RNA(antiapoptotic gene) weakened, and cattle encephalon Glycoside and ignotin injection can prevent these changes.Conclusions: 1 Bilateral cochlear damage is a stable technique to build an auditory deprivation animal models. 2 Three genes of Bcl-2, Bax and Fas express in the auditory cortex3 Bcl-2, Bax and Fas are involved in neuronal apoptosis process in auditory cortex after auditory deprivation. 4 Cattle encephalon glycoside and ignotin injection has protective effects on auditory center injury induced by auditory deprivation through inhibiting apoptosis pathway.