Chronological Changes Of The Blood-brain Barrier Dysfunction Were Involved To Neuronal Death In The Mice Hippocampal CA3 Region Of Initial Phase Of Epilepsy Induced By Kainic Acid

Objective:Epilepsy is the sudden abnormal discharge of neurons that can lead to chronic occurrences of transient cerebral dysfunction.The kainic acid(KA)-induced epilepsy experimental model is widely used to study the mechanisms underlying this disorder.Recently,the blood-brain barrier(BBB)has become an innovative alternative treatment target for epilepsy patients.KA causes neuronal injury and BBB damage in this experimental epilepsy model but the mechanisms underlying epilepsy-related neuronal injury,autophagy,and BBB damage remain unclear.Therefore,the present study investigated the relationships among neuronal injury,the expressions of autophagy-related proteins,and changes in BBB-related proteins during the acute phase of epilepsy to further understand the mechanisms and pharmacotherapy of epilepsy.Methods:A solution of KA and saline was stereotaxically administered into the unilateral ventricle(-1.0 mm,-0.22 mm,and-0.3 mm relative to bregma)of each mouse.Prior to surgery,all mice were anesthetized with 10%chloral hydrate(Aladdin,China)and placed into the stereotaxic apparatus.Subsequently,the animals were randomly divided into seven groups(n =14 each;7 animals for histological analyses and 7 animals for biochemical analyses)as follows:normal saline group(control-group),3?g/kg kainic acid group(3?g/kg KA-group),10 ?g/kg kainic acid group(10?g/kg KA-group).Normal saline group(control-group),3?.g/kg kainic acid group(3?.g/kg KA-group),10 ?g/kg kainic acid grop were sacrificed after 3 days.Following the KA injection,the 10KA group was divided into four subgroups(n = 14 in each group;7 animals used for histological analyses and 7 animals used for biochemical analyses)based on time:6 h,24 h,48 h,and 72 h.After the injection,all animals were returned to their cages where seizure severity was assessed in 10 min intervals for up to 2 h using the modified Racine scale:stage 0,normal behavior;stage 1,immobility;stage 2,forelimb and/or tail extension,rigid posture;stage 3,repetitive movements,head bobbing;stage 4,rearing and falling;stage 5,continuous rearing and falling;stage 6,tonic seizure or death.Immunohistochemistry was used to detect NeuN-labeled neuronal nuclear antigen,FJB-labeled neurons and Ibal-labeled microglia,GFAP-labeled astrocytes,PECAM-1 labeled vascular endothelial cells,ZO-1 labeled Tight junction protein changes,and the expression of LC3?/I,Beclin-1,Claudin 5 and ZO-1 were detected by Western blotting.Results:(1)At 72 h after KA treatment,the mice in the 10?g/kg KA group had obvious epileptic seizures,and obvious neuronal damage was seen in the hippocampal CA3 region.(2)The expression of Beclin-1 was gradually increasing from control,6 to 48h after KA injection.However,there was a small decrease at 72 h after the KA injection compared to 48 h after the KA injection.LC3?/LC3? levels in the hippocampus exhibited an increase at 6 h after the KA injection compared to the control group and peaked at 24 h;the expressions gradually decreased at 48 and 72 h.(3)The degree of Iba-1 immunoreactivity and the number of activated microglia cells in the 10?g/kg KA-group significantly increased in the hippocampal CA3 region compared to the control and 3?g/kg KA-groups.In the 3?g/kg KA-and 10?g/kg KA-groups,the activation and immunoreactivity of GFAP+ cells significantly increased compared to the control group.(4)PECAM-1 immunoreactivity was peaky increased in the hippocampal CA3 region at 24 and 48h after the KA injection.At 72 h after the KA injection,its immunoreactivity had somewhat decreased compared to at 48 h,but it remained higher than that of the control group.At 24,48,and 72 h after injection,the groups did not exhibit differences in ZO-1 immunoreactivity in the hippocampal CA3 region,but their immunoreactivities were significantly higher than those in the control group.(5)At 24,48,and 72 h after injection,the expressions of Claudin-5 were significantly higher than that of the control-group.ZO-1 protein levels in the hippocampus gradually increased from the control-group to the other groups at 6 h and 72 after injection.Conclusion:(1)KA-induced neuronal injury in the hippocampus is likely associated with early chronological changes in Beclin-1 and LC3? levels,which are correlated with autophagic cell death.(2)It is likely that the astrocytic and microglial activations observed in the present KA-induced experimental epilepsy model were related to neuronal death and BBB failure.(3)Transient early increases in the expressions of TJ-associated proteins,such as ZO-1 and Claudin-5,and changes in PECAM-1 expression may be associated with neuronal injury in the hippocampal CA3 region following injections of KA.The experimental results indicate that KA-induced neuronal death in the hippocampal CA3 region is related to autophagy.In addition,the overexpression of astrocyte activation and early transient increases in the expressions PECAM-1,ZO-1,and Claudin-5 may be closely related to neuronal injury.However,we only assessed changes in autophagy,the BBB,and neuronal injury during the acute phase of epilepsy.Further studies are necessary to verify the roles of autophagy and BBB damage in neuronal cell death using drugs.These results will provide novel therapeutic targets for the treatment of epilepsy.