Potential Role Of TDP-43 In Secondary Brain Injury After Intracerebral Hemorrhage In Rats: Key Mechanisms Involving Its Nuclear Loss And Phosphorylation

Part I: TDP-43 protein levels and distribution changes after ICHObjective: To investigate the different time points after TDP-43 changes after ICH.Methods:In vivoexperiment,42 rats(44 rats were used,42 rats were survived after the surgery)were randomly assigned to 7 groups of 6 rats each,a sham group and 6 experimental groups arranged by time: 3h,6 h,12 h,24 h,48 h,72 h after Intracerebral hemorrhage(ICH).All the rats in the experiment were killed at the indicated time point after ICH.The brain cortex of 6 rats in each group was extracted and used for double immunofluorescence analysis and western blot analysis.Results: 1,In order to detect the expression of TDP-43 after ICH,we tested the protein level of TDP-43 in nuclear and cytoplasm of the cerebral cortex respectively.The results demonstrated that,compared with the sham group,the protein level of TDP-43 in the nuclear were repeated changing after ICH,reached the lowest point at 48 h(p<0.01),and then rebounded gradually.In the cytoplasm,compared with the sham group,the protein level of TDP-43 in the nuclear were increased significantly after ICH,reached the highest point at 48 h(p<0.01),and then reduced gradually.2,The immunofluorescence staininganalysis of protein sample in vivo and vitro further verified the ICH-induced increase in cytoplasm in the protein level of TDP-43(P<0.01)Conclusion: TDP-43 in the cerebral cortex transferred from nuclear to cytoplasm after ICH and peaked at 48 h.The phosphorylation level of TDP-43 reached the highest level in cytoplasm after ICH at 48 h.Part II: The effect of regulating TDP-43 on the secondary brain injury after ICHObjective: To investigate the distribution expression changes of TDP-43 after intervening the TDP-43 plasmid mutation which could inhibit the phosphorylation sites S409/410 after ICH.And the underlying key mechanisms of TDP-43 involve in second brain injury induced by ICH.Methods: In vivo experiment,84 rats(90 rats were used,84 rats were survived)were randomly divided into 7 groups(n = 12 per group): sham group,ICH group,ICH+ vector group,ICH+ TDP-43 plasmid group,ICH+ TDP-43 mutation group,ICH+ control RNA group,ICH+ TDP-43 si RNA group.After transfection of TDP-43 plasmid?TDP-43 mutation and TDP-43 si RNA at 48 h,we inject 80 ul autologous blood in the basal ganglia of rats.At 48 h after ICH,all the rats were killed.The brain cortex of 6 rats per group were extracted for terminal deoxynucleotidyl transferase-mediated d UTP nick end labeling(TUNEL)staining,fluoro-jade B(FJB)staining,and 6 rats per group were used for western blot analysis.The vitro experiment LDH was also performed to evaluate the cell death of neurons after intervention with TDP-43 mutation.Results: 1?The protein level of PITDP-43 increased significantly after ICH comparing with the sham group(p<0.05)and reduced significantly after intervening with TDP-43 mutation(p<0.05).2?After intervening with TDP-43 mutation,the protein level of TDP-43 reduced significantly in cytoplasm comparing with ICH group(p<0.05),and the protein level of TDP-43 in nuclear increased significantly comparing with ICH group(p<0.001).3?The TUNEL ?FJB staining and LDH analysis all indicated that intervening with TDP-43 mutation could significantly reduce the number of neuron apoptosis necrosis comparing with ICH/oxy Hb group(p<0.05).Conclusion:The phosphorylation level of TDP-43 reduced significantly after intervening TDP-43 mutation which could inhibit the phosphorylation site S409/410.It reduced the protein level of TDP-43 transferring from nuclear to cytoplasm which contribute to the nuclear loss of TDP-43 and mitigated the second brain injury induced by ICH.Part III: The study of TDP-43 in second brain injury induced by ICH.Objective: To investigate the phosphorylation level of TDP-43 affected by CN after ICH and the potential protection mechanism of inhibiting the TDP-43 phosphorylation sites S409/410 of TDP-43.Methods: In vitro experiment,calcineurin activity kit was used to test the activity of calcineurin after ICH and the intervention of CHA?FK506.The protein level changes of PITDP-43 were tested after ICH and the intervention of CHA?FK506.In vivo experiment,42 rats(45 rats were used,42 rats were survived)were randomly divided into 7 groups(n = 12 per group): sham group,ICH group,ICH+ vector group,ICH+ TDP-43 plasmid group,ICH+ TDP-43 mutation group,ICH+ control RNA group,ICH+ TDP-43 si RNA group.After transfection of TDP-43 plasmid?TDP-43 mutation and TDP-43 si RNA at 48 h,we inject 80 ul autologous blood in the basal ganglia of rats.At 48 h after ICH,all the rats were killed.The brain cortex of rats were extracted for western blot analysis.Result:1?The activity of CN increased significantly after ICH.CHA and FK506 could respectively increase and reduce the activity of CN.2?In vivo experiment,the protein level of m-TOR and DCTN1 increased significantly after intervening TDP-43 mutation comparing with ICH group(p<0.05).3?The level of autophagy increased significantly comparing with sham group(p<0.05)and reduced significantly after intervening with TDP-43 mutation(p<0.05).Conclusion: The activity of CN increased significantly after ICH which could promote the dephosphorylation of PITDP-43,and CHA?FK506 could respectively increase and reduce the activity of CN.After intervening with TDP-43 mutation which could inhibit phosphorylation site S409/410,the level of autophagy reduced significantly comparing with ICH group and reduced the second brain injury.