Protective And Repair Effects Of Neuritin Gene In Cerebral Ischemia-Reperfusion Injury

Object: In order to identify and screen transgenic positive mice that successfully overexpressed Neuritin,and to evaluate the neuroprotective and repairing effects of Neuritin protein in mice with cerebral ischemia-reperfusion injury.Methods:1.Identification of the integration and expression of Neuritin gene in transgenic mice(Nrn1-Tg).(1)Identification of the target gene at the genome level: The DNA of transgenic offspring mice were extracted and the mice that successfully integrated the inserted sequence were initially verified by PCR.The positive mice were re-identified by southern blot,and the mice that were positive for both tests were mated with wild-type mice(WT).(2)Identification of the target gene at the transcription level: qRT-PCR was used to detect the level of Neuritin mRNA in Nrn1-Tg and WT mice tail and hippocampal tissue.(3)Identification of the target gene at protein level: The expression of Neuritin in hippocampus of transgenic mice was detected by western blot.2.Transient global ischemia(TGI)was established by double occlusion of the common carotid arteries(CCAs).Thirty-six C57BL6 mice were divided into transgenic model group(Tg-TGI),wild type model group(WT-TGI),and sham operation group(sham).Sham-operated animals were treated identically,except for the occlusion of both CCAs,n = 12.Each group was divided into four subgroups,namely,1,3,7,and 14 days(n = 3).The mice were sacrificed on day 1,3,7,and 14 days after reperfusion to obtain brain tissue.3.The temporal expression profile of Neuritin in hippocampal after TGI was detected by qRT-PCR,western blot and immunohistochemistry.4.To evaluate the effects of over-expressed Neuritin on neuronal injury and apoptosis after TGI.(1)Nissl staining was used to evaluate the degree of damage to hippocampal CA1 neurons after TGI.(2)The expression of cleaved PARP1 in hippocampal CA1 region was detected by immunohistochemistry.(3)western blot was used to detect the expression of cleaved PARP1 in hippocampal tissue after TGI.5.To detect the effects of Neuritin on the ability of regenerate and repair of neurons after TGI.(1)The expression of nerve regeneration markers(Growth associated protein-43,GAP-43;synaptophysin,SYN-38;neurofilament protein,NF-200)in hippocampal CA1 region were detected by Immunohistochemical staining.(2)The expression levels of GAP-43,SYN-38,and NF-200 in hippocampus were detected by western blot.6.To evaluate the effects of Neuritin on spatial learning and memory ability after TGI.Escape latency and numbers of crossing platforms of Nrn1-Tg mice and WT mice after TGI were detected by the Morris Water Maze(MWM)test.Results: 1.The transgenic positive mice that over expressing Neuritin were identified successfully.(1)Identification of the target gene at the genome level: the integrative mice screened by PCR are consistent with the identification results by southern blot,with 1-2 copy numbers.A total of 145 mice were bred,including 39 Nrn1-Tg mice.(2)Identification of the target gene at the transcription level: qRT-PCR results showed that compared with WT mice,the level of Neuritin mRNA in tail tissue and hippocampus of Nrn1-Tg mice was significantly increased(p<0.05).(3)Identification of the target gene at protein level: WB results showed that compared with WT mice,the expression of Neuritin protein in the hippocampus of Nrn1-Tg mice was significantly increased(p<0.05).2.After the TGI,the expression of Neuritin mRNA gradually increased on the first day,reached its peak on the seventh day,and fell back on the fourteenth day.And on the 1st,3rd,and 7th days,the level of Neuritin mRNA in Tg-TGI group was significantly higher than that in WT-TGI group(p<0.05).Results of WB and immunohistochemistry showed that after TGI,the expression of Neuritin protein in hippocampal tissue and CA1 area gradually increased on the first day then fell back to the first day level after reaching the peak on the 3rd day.Compared with WT-TGI mice,the expression of Neuritin protein in the Tg-TGI group was significantly increased on days 3,and 7(p<0.05).3.Neuritin can reduce nerve cell damage and save damaged cells from apoptosis induced by TGI.(1)The results of Nissl staining showed that in the sham group,the nerve cells of hippocampal CA1 area were closely and neatly.The number of nerve cells of TGI group decreased,and the neurons showed contracted cell body and constricted nucleus,which suggested that the modeling was successful.Compared with WT-TGI,the number of damaged neurons in the hippocampal CA1 region of the Tg-TGI group reduced significantly on days 3 and 7(p<0.05).On day 14,nerve cell counts returned to no difference between groups.(2)Results of WB and immunohistochemistry showed that in the first week after TGI,compared with the WT-TGI group,the expression levels of cleaved PARP1 protein hippocampus of Tg-TGI group were significantly reduced(p<0.05).There were no differences between groups on day 14.4.Neuritin can enhance the regeneration and repair ability of nerve cells after TGI.(1)The expression of GAP-43 protein gradually increased on the first day after TGI,and decreased after reaching the peak on the third day.The protein expression of GAP-43 in the Tg-TGI group was significantly higher than that in the WT-TGI group on days 3 and 7(p<0.05).(2)The expression of SYN-38 protein gradually increased on the first day,reached its peak on the seventh day,and then fell back on the fourteenth day.The peaks of the Tg-TGI group on the third and seventh days were significantly higher than those of the WT-TGI group(p<0.05),the expression level in the WT-TGI group was higher than that in the Tg-TGI group on the 14 th day.(3)Compared with WT-TGI,the expression of NF-200 protein in the Tg-TGI group increased significantly on days 1-7(p<0.05),and there was no difference between the groups on day 14.5.Neuritin improved spatial learning and memory ability of transgenic mice after TGI.(1)The results of the MWM test showed that the escape latency of the three groups gradually shortened as the number of training sessions increased.Compared with the WT-TGI group,the escape latency of the Tg-TGI group was significantly shortened on days 3,4,and 5(p<0.05).(2)In the space exploration experiment,the times of crossing the platform and the exploration time in the target quadrant of Tg-TGI group increased significantly compared to the WT-TGI group(p<0.05).(3)The swimming exploration path also revealed that Tg-TGI mice have a more targeted quadrant search strategy,while WT-TGI mice tend to swim randomly.Conclusion: Transgenic positive mice of overexpressing Neuritin were screened and identified successfully.Overexpression of Neuritin can reduce the damage of nerve cells,and can reduce the apoptosis of nerve cells by inhibiting the degradation of PARP1 protein,and accelerate the recovery of nerve cells in the CA1 region after TGI.In addition,during the critical period of axon regeneration after injury,up-regulating Neuritin can significantly increase the expression of NF200,GAP-43 and SYN-38 proteins,which provide a favorable microenvironment for the regeneration and remodeling of neurites,and effectively improve the ability of nerve regeneration and repair after TGI,accelerating the improvement of spatial learning and memory ability of mice after TGI.