Role Of Lingo-1 In The Formation Of Hippocampal Aberrant Neural Circuits After Status Convulsion In Immature And Adult Rats

PART I: ALTERATION OF HIPPOCAMPAL NEURAL CIRCUITS FOLLOWING STATUS CONVULSION IN IMMATURE AND ADULT RATSObjective: To investigate the alteration of hippocampal myelin sheaths,axons and synapses after status convulsion(SC)in immature and adult rats.Methods: Both immature(postnatal day 21 [P21])and adult(postnatal day 60 [P60])Sprague–Dawley(SD)rats were used and were respectively divided into two groups,the control group and the SC group.Rats in the SC group were induced SC by lithium-pilocarpine.Then transmission electron microscopy(TEM)was performed to observe the structure alterations of myelin sheath and synapses in hippocampi.Western blotting was used to detect the expression of myelin basic protein(MBP),Neurite outgrowth inhibitor protein A(Nogo A),oligodendrocyte myelinglycoprotein(OMgp),myelin associated glycoprotein(MAG),synaptophysin(SYP),and the four AMPA recepor subunits.At last the Lingo-1 expression was tested by immunofluorescence staining and western blotting to explore whether Lingo-1 was related with the brain damages induced by SC.Results:(1)The results showed that the myelin sheath in hippocampi presented stratification,vacuolation,collapse,and disruption after SC.The thickness of myelin was decreased,accompanied by down-regulation of MBP expression in immature and adult rats' hippocampi after SC.(2)The expression of NF200 in immature and adult rats' hippocampi was decreased20 days after SC.The expression of three major myelin-derived inhibitors(Nogo A,OMgp,and MAG)were increased in immature and adult rats with different degree at different time points after SC.(3)The number of synapses and the thickness of postsynaptic density(PSD)in hippocampal CA1 region was decreased,but the length of active zone(AZ)in presynaptic membrane was increased.The expression of SYP in immature rats' hippocampi was decreased at the 1st day after SC,but increased at the20 th and 60 th day after SC.While the expression of SYP in adult rats' hippocampi was decreased from the 1st day lasting to 60 th day after SC.(4)The Glu R1/4 subunits were predominantly expressed in immature rats' hippocampi,and Glu R1/3 subunits in adult rats' hippocampithe.The expression of Glu R2 subunit in immature and adult rats' hippocampi weredecreased after SC.(5)The expression of Lingo-1 in immature and adult rats' hippocampi were increased from the 5th day lasting to 60 th day after SC.Conclusion: After SC,the myelin sheath,axons and synapses in immature and adult rats' hippocampi were damaged.The increased expression of Lingo-1 may be involved in the pathological process of hippocampal circuit damage.Furthermore,the severity of hippocampi damage in immature and adult rats were different.The immature rats were less vulnerable to seizure-induced damage to hippocampi.PART II: CONSTRUCTION AND TESTING OF LENTIVIRAL VECTORS ENCODING LINGO-1 SHRNAObjective: To construct lentiviral vectors encoding Lingo-1 shRNA with silencing or over-expression efficiency,and to verify the efficiency of lentiviral vectors after injected into rats' brain by intracerebroventricular(ICV)injection.Methods: The two kinds of lentiviral vectors were injected into the cerebroventricular of immature and adult rats.The green fluorescent protein(GFP)expressed by lentiviral vectors were observed in the lateral ventricles of hippocampal sections using fluorescence microscopy.The Lingo-1 expression in hippocampi was tested by western blotting to make sure the optimal injection dosage and the efficiency of the two kinds of lentiviral vectors.Results:(1)The results showed GFP expressed by lentivirus was observed in the lateral ventricles of hippocampal after ICV injection with lentiviral vectors,which could be expressed in brain lasting at least 28 days.(2)The optimal dosage of lentiviral vectors to up-regulating(down-regulating)Lingo-1 expression for immature rats was 5?l(1.5?l),for adult rats was 8?l(3?l).(3)After lentivirus administration by ICV injection,the Lingo-1 expression of hippocampi was increased or decreased significantly at the 7th day after injection,and lasting to the 28 thday post ICV injection.Conclusion: The two kinds of lentiviral vectors could be effective after being injected into brain through ICV injection.PART III: ALTERATION OF HIPPOCAMPAL NEURAL CIRCUITS FOLLOWING STATUS CONVULSION AND REGULATING LINGO-1 EXPRESSION WITH RNA INTERFERENCE IN IMMATURE AND ADULT RATSObjective: To investigate whether up-or down-regulating Lingo-1expression with RNA interference cloud affect the alteration of hippocampal myelin sheaths,axons and synapses caused by SC in immature and adult rats.Methods: These rats used in the Part I were the NI group without RNA interference.Then another immature and adult SD rats were selected and randomly assigned to three groups [LV,OLV,NC(administrated with negative control virus)] depending on being injected with which kind of lentiviral vector.Next each group divided into two subgroups,one subgroup was as the control group without SC,the other one was SC group treated with lithium-pilocarpine.TEM was performed to observe the structure alterations of myelin sheath and synapses in hippocampi.Western blotting was used to detect the expression of MBP,Nogo A,OMgp,MAG,SYP and the Glu R2 subunits.Timm stain was performed to observe the Mossy fiber sprouting(MFS)in the hippocampi CA3 region of immature and adult rats.At last electroencephalogram(EEG)monitoring was used to record electrophysiological activities after SC.Results:(1)The incubation of inducing SC with lithium-pilocarpine had no significant differences among the four groups.(2)Down-regulating Lingo-1 expression could ameliorate the SC-induced damages to myelin sheath,increase the thickness of myelin and the expression of MBP.While up-regulating Lingo-1 expression had opposite effects.(3)Down-regulating Lingo-1 expression could reduce the MFS in hippocampal CA3 region and reduce the expression of myelin-derived inhibitors(Nogo A,OMgp,and MAG).Up-regulating Lingo-1 expression could not ameliorate the SC-induced damages to axons.(3)Down-regulating Lingo-1 expression could ameliorate the SC-induced damages to the structure of synapses.Up-regulating Lingo-1 expression had the opposite effects.(4)Down-regulating Lingo-1 expression could inhibit the expression of Glu R2 subunit in immature rats' hippocampi,but increase the expression of Glu R2 subunit in adult rats' hippocampi.After up-regulating Lingo-1 expression,the expression of Glu R2 subunit in immature rats' hippocampi was increased 60 days after SC,but which was decreased in adult rats' hippocampi.(5)The EEG recording showed that there were no significant differences among the groups with/without different virus administration about the incidence of EEG discharges during the chronic period after SC;but the severity of EEG discharges were significantly reduced after down-regulating Lingo-1 expression.Conclusion: Down-regulating Lingo-1 expression could amelioratethe SC-induced damages to myelin sheath,axons and synapses in immature and adult rats' hippocampi.Up-regulating Lingo-1 expression had the opposite effects.Down-regulating Lingo-1 expression could reduce the severity of EEG discharges during the chronic period after SC.