| Objective: 1.To observe the apoptosis-related marker proteins of hippocampal neurons in radiation-induced brain injury mice: including activation of Caspase-3,AIF and DNA fragmentation,to explore the protective effect on neuronal apoptosis in hippocampus of electroacupuncture in radiation-induced brain injury mice.2.To observe the ultrastructure of brain tissue in mice with radiation-induced brain injury;detect changes of neurons,synapse morphology,synapsin-? and mRNA expression of hippocampal,to discuss the protective mechanism of electro-acupuncture at "Baihui","Fengfu" and "Shenshu" on synaptic plasticity in radiation-induced brain injury mice.Methods: 4-week-old male C57 BL / 6J mice,80 were randamly divided into 8 groups,10 in each group.By using of 8Gy radiation exposure to made radioactive brain injury mice model,and EA treatment was given in the second day.After the end of EA,each mice were deeply anesthetized with 2% sodium pentobarbital(0.1ml / 10g),open the chest to expose the heart and perform perfusion with 0.1mol / L of PBS solution(PH7.4)30ml rinse vascular perfusion about 10 min,get brains quickly onbed of ice,separated the portion hippocampus.IHC and Transmission electron microscopy detecting neuronal apoptosis and synaptic ultrastructural changes.To detect synaptic protein and its mRNA expression changes in hippocampal neurons by WB and Q-PCR.Results: 1.The immunohistochemical method for the detection of TUNEL,AIF,and Caspase-3 showed that the IOD values of TUNEL,AIF and Caspase-3 positive cells in the irradiation model group were significantly higher than those in the blank group,suggesting an increase in hippocampal apoptotic cells.The difference was statistically significant(P<0.01,P<0.001,P<0.05).Compared with radiation irradiation group,the IOD of AIF and Caspase-3 positive cells in the EA 1W group was significantly decreased(P<0.01,P<0.05);in EA 2W group,the IOD value of AIF positive cells was significantly decreased(P<0.01);furthermore,the IOD of TUNEL,AIF,and Caspase-3 positive cells in the EA 3W group were significantly decreased,and the difference was statistically significant(P<0.05,P<0.001,P<0.05).2.Results of nucleus gap nucleus to cytoplasm ratio: The nuclear gap of irradiation model group was significantly decreased but the nucleus to cytoplasm ratio was significantly upraised(P<0.001,P<0.01).To the radiation irradiation group,the nucleus gap of EA1 W & EA 2W group and the nucleus to cytoplasm ratio in EA 1W were no significant changes;the nucleus gap of EA 3W group and the nucleus to cytoplasm ratio in EA 2W & EA 3W were significantly increased(P<0.01,P<0.05,P<0.01).There was no significant changes in the nucleus gap and the nucleus to cytoplasm ratio of all the sham-EA groups.3.Synapse cleft and PSD: Synaptic cleft and PSD of radiation irradiation group were decreased than blank group(P<0.01,P<0.05).To with the radiation irradiation group,the synaptic cleft of EA 1W & 2W and the PSD of EA 1W group had no significantly changes;the synaptic cleft of EA 3W and the PSD of EA 1W & 2W had a significant increased(P<0.001,P<0.001,P<0.01);but all the sham-EA group's synaptic cleft and PSD thickness showed no statistically significant changes.4.The results of mitochondrial ultrastructure statistics: Compared with the control group,the mitochondrial numerical density & surface density & volume density & specific surface area in the hippocampus of the irradiated group were significantly decreased(P<0.05,P<0.001,P<0.01,P<0.01).Compared with the irradiated irradiation group,the mitochondrial numerical density & surface density & volume density & specific surface area didn't have a specific differences in EA 1W;the numerical density & surface density & volume density were also no changes while the specific surface area increased significantly in EA 2W mice(P<0.05);In the same time,the mitochondrial surface density & volume density & specific surface area were significantly upraised in EA 3W group(P<0.01,P<0.01,P<0.01);but these indexes were no statistically significant change in all of sham-EA group.5.The results of synapsin 1 mRNA expression in hippocampus: The expression of synapsin ? mRNA in the hippocampus of the radiation-induced model mice was declined than blank mice(P<0.05).Compared with the radiation-induced group,the expression of synapsin ? mRNA in the hippocampus of EA 1W group had no statistical significance.The expressions of synapsin 1 mRNA in hippocampus of EA 2W and EA 3W group were significantly increased(P<0.001,P<0.001).The expression of synapsin ? mRNA in the hippocampus of sham-EA 1W d 2W were dclined(P<0.01,P<0.01),but not in the sham-EA 3W group.Compared with sham-EA 1W group,the expression of synapsin ? mRNA in hippocampus did not change significantly in EA 1W group.Compared with sham-EA 2W group,the expression of synapsin ? mRNA expression increased in EA 2W group(P<0.01).Compared with sham-EA 3W group,the expression of synapsin ? mRNA in hippocampus of EA 3W group was significantly increased(P<0.001).6.Western blot analysis of synapsin ? protein in hippocampus: The expression of synapsin ? protein in the hippocampus was significantly decreased in the radiation group(P<0.05).For the radiation group,the protein expression of synapsin ? in the hippocampus of EA 1W,EA 2W and EA 3W groups were all significantly increased(P<0.05,P<0.01,P<0.01).The protein expression of synapsin ? in the hippocampus of sham-EA 1W group was significantly increased(P<0.001).Compared with sham-EA 1W group,the expression of synapsin ? protein in hippocampus of EA 1W group was significantly lower(P<0.05).Compared with sham-EA 2W group,the expression of synapsin ? protein in the hippocampus of EA 2W group had no statistical difference.Compared with sham-EA 3W,the expression of synapsin ? protein in hippocampus of EA 3W group had no statistical difference.Conclusion: 1.Electroacupuncture on “Baihui”,“Fengfu” and “Shenshu” can effectively reduce the expression of neuronal apoptosis inducing factor and apoptotic protein in the hippocampus of mice with radiation-induced brain injury,and reduce the damage to cell DNA caused by radiation.Antagonize radiation exposure to apoptosis after brain injury.2.Electroacupuncture can promote hippocampal synaptic plasticity in mice with radiation brain injury.The mechanism may be to improve the ultrastructure of neurons,synapses and mitochondria in the hippocampus,and upregulate the expression of synaptophysin I mRNA and its protein. |
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