Role Of RhoA/ROCK Signal Pathway In The Formation Of Hippacampal Axonal Regeneration After Status Convulsion In Brain

Objective: To explore dynamic changes of axon growth inhibitory factors(Nogo A,MAG,OMgp(MOG)),transmembrane protein Lingo-1,and Rho A in hippocampus of rats at different times of epilepsy after SC.Methods: Initially,to determine the effects of SC,the protein expression levels of Nogo A,MAG,OMgp,Rho A,Lingo-1 were measured by western-blot and immunofluorescence,and RT-PCR for the m RNA expression of Lingo-1.In the study,120 rats selected were evenly divided into control group(24 rats)and SC group(96 rats).SC model was established by Li Cl-Pilo method.Methods: 120 selected adult SD rats were divided into control group(24 rats)and SC group(96 rats).96 rats of SC group were administered with Lic1-Pilo by intraperitoneal injection to induce Status Convulsion(SC).At different times of SC,Immunofluorescent staining method was applied to determine the protein expression levels of Nogo A,MAG,OMgp,Rho A,and Lingo-1;Western blot was used to measure dynamic changes of Nogo A,MAG,OMgp,Rho A,Lingo-1,and p-Rho A content;RT-PCR was taken to detect the m RNA expression level of transmembrane protein Lingo-1 in hippocampus.Results:(1)The changes of axon growth inhibitory factors:Nogo A,MAG,OMgp in hippocampus of rats all increased during the course of epilepsy after SC,but exhibiting various time-sequential dynamic changes.MAG: On the 5th day of SC,the expression increased(P < 0.01),then declined slowly.On the 20 th day and the 60 th day of SC,it had no statistical significance compared with control group.The results by western blot were slightly different from those by Immunofluorescent test,which expressions were higher than normal level on the 5th day and the 20 th day of SC(P<0.001,P<0.01),and declined to normal on the 60 th day of SC.Nogo A: On the first day and the 5th day of SC,there were not significant changes compared with the normal group.On the 20 th day,it rose up(P < 0.01).On the 60 th day,no statistical difference had been observed compared with the normal group.The results by western blot were similar to those by Immunofluorescent test.OMgp: It wasn't markedly elevated on the first day,it increased on the 20 th day and the 60 th day(P<0.01,p<0.001).The results by western blot were similar to those by Immunofluorescent test.(2)Lingo-1 tests showed that expressions increased gradually on both protein level and genetic level(m RNA)after SC,and started to slide after their peaks.The genetic expression was prior to protein expression.On protein level,the expression had no statistical significance compared with the normal on the first day.It reached its peak on the 5th day of SC(p < 0.001),and then decreased gradually on the 20 th day(p<0.05).It returned back to normal on the 60 th day;By semi-quantitative PCR,results showed that expression level of Lingo-1 m RNA had reached its peak(P<0.001)quickly on the first day of SC,and remained high(P<0.001)on the 5th day and the 20 th day.It didn't drop back to normal on the 60 th day.(3)Rho A: Compared with the normal group,difference was not statistically significant.The total amount of Rho A hadn't shown obvious change after SC.Phosphorylation Rho A(p-Rho A): It rose up quickly after SC(p < 0.001),reached its peak(p < 0.001)on the 5th day,and fell down(p < 0.001)on the 20 th day.Compared with the normal,there was no statistical difference on the 60 th day.Conclusion:(1)All though the changing directions for Myelin-associated axon growth inhibitory factors are not exactly the same after SC,all of them increase in different periods.Therefore,their combined forces are associated with Phosphorylation Rho A on Rho A/ROCK signaling pathway.(2)The Lingo-1 and phosphorylation Rho A present an increased tendency after SC,and then decline gradually during the chronic phase.The change directions and times of Lingo-1 and phosphorylation Rho A are similar,which points out that Rho A phosphorylation is largely mediated by Lingo-1 and Rho A/ROCK signaling pathway is activated after convulsion,eventually leading to the collapse of neurons axon growth cone and further to the impairment of axonal function.Objective: In part I,our research have found that Lingo-1expressions(on both gene and protein)increase after SC and gradually decline with the extension of time of epilepsy.In experiments of this part,we want to use the sh RNA fragments transfection to inhibit the expression of Lingo-1 protein,aiming to understand whether LV transfection can reduce Lingo-1 expression of target cells and tissues.Also we want to know whether it would be fit the requirements of the next experiments.Method: Three different LV Lingo-1 sh RNA fragments(SC+LV LINGO-1/SHRNA 3086,SC+LV LINGO-1/SHRNA 3087,SC+LV LINGO-1/SHRNA 3088)were designed and packaged to generate the experimentrequired LV Lingo-1 / sh RNA fragments by using GV118 as the carrier.These designed Lingo-1/sh RNA fragments were verified respectively on transfection cell and animal levels.(1)on the cellular level: the cultured C6 cells were transfected with LV Lingo-1/sh RNA.The changes of Lingo-1 expressions were verified through immunofluorescence and PCR.(2)On the animal level: 112 selected SD rats were divided into control groups(32 rats)and SC model groups(80 rats).Control groups had normal group and normal + lentivirus empty virus group;SC model groups included SC group,SC + empty virus group,SC + LV Lingo-1 /sh RNA 3086,SC + LV Lingo-1/ sh RNA 3087,SC + LV Lingo-1 / sh RNA 3088.There were 7 groups in total and each group had 16 rats.Through lateral ventricle stereotaxic,the sh RNA fragments were injected into lateral ventricle(the group expected to be executed on the 59 th day would be injected with LV LINGO-1/SHRNA once again on the 30 th day).On the 10 th day and 59 th day after the injection of LV Lingo-1/sh RNA,animals were executed to study Lingo-1 protein expression by Western blot.Results:(1)On protein and cellular level,three kinds of designed LV Lingo-1/sh RNAs were able to inhibit protein expressions of Lingo-1 in cultured C6 cells,among which fragment 3087 was the most effective.(2)Animal experiments also showed that whether on the 10 th day or the 59 th day after injection,three kinds of designed LV Lingo-1/sh RNAs were able to inhibit protein expressions of Lingo-1,and inhibition by LV Lingo-1/sh RNA 3087 was the most significant.Conclusion: The packaged LV Lingo-1/sh RNA fragments can definitely inhibit the expression of Lingo-1,among which fragment 3087 is the most effective.Objective: To understand the effects on susceptibility to convulsion,cognitive function for rat after SC,as well as its pathological basis by blocking phosphorylation of Rho A and inhibiting expression of Lingo-1.Method: SC animal model was established through LiCl-Pilo,by which SD rats of 60 days were divided into HF(Fasudil hydrochloride)intervention group(156 rats)and LV Lingo-1/sh RNA intervention group(195 rats).LV Lingo-1/sh RNA intervention group included LV Lingo-1/sh RNA intervention group and LV Lingo-1/sh RNA+HF intervention group.For these two groups,on the next day after model had been established,the stereotaxic instrument was used to locate the lateral ventricle and each rat was injected with 5?l of LV Lingo-1/sh RNA through the lateral ventricle.Meanwhile,from the first day after SC,LV Lingo-1/sh RNA+HF intervention group had received intraperitoneal injection of HF(10 mg/kg ×30 days).30 days after intraperitoneal injection,both groups were injected with the similar dosage of LV Lingo-1/sh RNA again;In the mean time,the normal group was established,with normal + LV Lingo-1/sh RNA group being as negative control.From the first day after SC,HF intervention group had received intraperitoneal injection of HF(10 mg/kg ×30 days).Meanwhile,the normal group was established and normal + HF group was set as negative control.On the 59 th day after intervention(the 60 th day for SC),Water maze experiment and the new object recognition experiment were adopted to test animal learning and cognitive function;Li Cl-Pilo was used to induce convulsion attack again to study changes of convulsion susceptibility for rats;HE staining and Nissl's staining were applied for us to determine pathological changes of brain tissues;Western blot was employed to detect MBP protein expressions,thus to understand changes in myelin sheath for SC rats after various intervention.ocking phosphorylation of RhoA and inhibiting expression of Lingo-1.Results:(1)The change of cognitive function: Water maze experiment showed that in the obvious platform experiment on the first day,time and distance for finding platform were not statistically significant among 5 groups of rats;with the extension of the training time from day 2 to day 5,time to find the platform for each group were getting shorter.On the 6th day,the obvious platform experiment indicated that for LV Lingo-1 /sh RNA intervention group,the times of finding platform by SC+LV Lingo-1 /sh RNA intervention group and the SC+LV Lingo-1 / sh RNA + HF intervention group were much more than for SC group,but still less than normal group,which was statistically significant(P<0.001,normal group vs.SC intervention group;p<0.01,control group vs.SC intervention group,SC+LV Lingo-1/sh RNA intervention group vs.SC intervention group,SC+LV Lingo-1/sh RNA+HF intervention group vs.SC intervention group);(2)for HF intervention group,the times of finding platform by SC+HF intervention group were much more than by SC intervention group,which was statistically significant(P<0.05,normal group vs.SC intervention group,control group vs.SC intervention group,HF intervention group vs.SC intervention group).New object recognition experiments suggested:(1)For the LV Lingo-1 / sh RNA intervention group,the experiment determined that LV Lingo-1 / sh RNA intervention had effects on the abilities for SC rats to explore new object(being expressed in preference index: i.e.,time for recognizing new object/ time for recognizing old object,comparison of ratios group by group).The abilities for SC group to explore new object were much lower than for normal group and the control group,the differences were statistically significant(P < 0.05,SC group vs.normal group,SC group vs.negative control group).However,the abilities for LV Lingo-1 / sh RNA intervention group and the LV Lingo-1/sh RNA+HF intervention group increased in evidence compared with SC group,and the differences were statistically significant(P<0.05,LV Lingo-1/sh RNA intervention group vs.SC group,and LV Lingo-1/ sh RNA + HF intervention group vs.SC group).The situation is similar for the ability to explore space,the difference between normal group and SC group was even greater(P<0.01),and the joint interventions showed more positive efficacy(P<0.01,SC group vs.normal group and LV Lingo-1/ sh RNA + HF intervention group vs.SC group;P<0.05,SC group vs.negative control group and LV Lingo-1/sh RNA intervention group vs.SC group).The results above indicated that Lingo-1/ sh RNA transplanting can improve rats' abilities to explore new object in SC model.(2)For the HF intervention group,the experiment determined that HF intervention had effects on rats' abilities to explore new object in SC model(being expressed in preference index).The abilities for SC group to explore new object were much lower than for normal group and control group,the differences were statistically significant(P < 0.01,SC group vs.normal group;P<0.05,SC group vs.control group and HF group vs.SC group).The preference indexes for exploring space were similar to for exploring new object(P<0.05,SC group vs.normal group,SC group vs.control group,HF group vs.SC group).The results above indicated that it could also improve rats' abilities to explore new object in SC model by independently using HF.(2)The change of Convulsions susceptibility:(1)Li Cl-Pilo was injected again 60 days after SC to induce convulsion attack to study changes of convulsion susceptibility under various interventions by comparing convulsion latencies group by group.(1)For LV Lingo-1/sh RNA intervention group,comparing normal group with control group,the convulsion latency for SC group was reduced evidently,and the differences were statistically significant(P < 0.01,SC group vs.normal group;P < 0.05,SC group vs.control group);After Lingo-1/sh RNA intervention,the convulsion latencies for SC rats were prolonged obviously,the differences were statistically significant(P<0.01,SC group vs.LV Lingo-1/sh RNA intervention group;p<0.001,SC group vs.LV Lingo-1/sh RNA+HF intervention group).The experiment suggested that LV Lingo-1 sh RNA could decrease the convulsions susceptibility of SC rats.(2)For the HF intervention group,comparing normal group with control group,the convulsion latencies for SC rats were reduced obviously,the differences were statistically significant(P<0.001,SC group vs.normal group;SC group vs.control group);After HF intervention,the convulsion latencies were increased evidently,and the differences were statistically significant(P<0.05,SC group vs.HF intervention group).The results suggested that HF could lower convulsions susceptibility of SC rats.(3)The change of MBP protein expression in hippocampus of SD rat:(1)For LV Lingo-1/sh RNA intervention group,Western-blot detection suggested that MBP protein for SC group was remarkably lower than for normal group and control group,the differences were statistically significant(P<0.001,SC group vs.normal group;SC group vs.control group);After LV Lingo-1/sh RNA fragment intervention,MBP content increased remarkably,the differences were statistically significant(P<0.001,SC group vs.LV Lingo-1/sh RNA intervention group,SC group vs.LV Lingo-1/sh RNA intervention +HF group).(2)For HF intervention group,Western-blot detection suggested that MBP protein for SC group was remarkably lower than for normal group and control group,the differences were statistically significant(P<0.001,SC group vs.normal group;SC group vs.control group);After HF intervention,its expression obviously exceed SC group's,the differences were statistically significant(P<0.001,SC group vs.HF group).(4)The pathological change of brain tissue: HE staining indicated that after SC,in hippocampal CA1,CA3 areas,pyramidal neurons had become dark and pyknotic,the structure was disorder,the number was decreased,and the neuronal cells were liquefied;After LV Lingo-1/sh RNA and LV Lingo-1/sh RNA +HF intervention,hippocampal structure recovered,cell morphology improved somewhat,the number of cells increased compared with SC group,but it still didn't recover back to normal condition.Further observing the changes of Nissl's body by Nissl's staining,the results were similar to those by HE staining,that is,in hippocampal CA1,CA3 areas after SC,the defection of Nissl's bodies was obvious,the structure was arranged irregularly,and the number declined;After LV Lingo-1/sh RNA and LV Lingo-1/sh RNA +HF intervention,Nissl's bodies increased compare to SC group,hippocampal structure recovered,but it still didn't recover back to normal condition.Conclusion: The cognitive behavioral experiments also show that they would be helpful to improve cognitive learning function after SC and prolong the convulsion latency In pathology,both HF and Lingo-1silence can repair the damages to the hippocampus structure and cells,ease the defections of Nissl bodies after SC,as well as can restore the injured myelin,which indicates that HF and Lingo-1 silence have some effects on restoring the damaged hippocampal structure and neurons after SC.