The Role Of N-methyl-D-aspartate Receptors In Epilepsy-induced Axonal Impairment And Its Molecular Mechanism

Part One Axonal impairment in brain tissues of refractory epilepsypatients and pentylenetetrazole-kindled ratsObjectives: To identify the characteristics of axonal impairment in brain tissues of epilepsy patients and pentylenetetrazole(PTZ)-kindled rats.Methods: Brain tissues resected from refractory epilepsy patients and white matters separated from PTZ-kindled rats were collected.The level of neurofilament heavy(NF-H),amyloid precursor protein(APP),total tau protein,phosphorylated tau S396(p-Tau S396)and phosphorylated tau T231(p-Tau T231)in brain tissues of refractory epilepsy patients and white matters of rats kindled with PTZ for 1day,1 week,2 weeks,3 weeks and 4 weeks were measured using western blot.And the relative phosphorylation level of tau was calculated.Results: In brain tissues of refractory epilepsy patients,NF-H and total tau level were significantly decreased compared to controls,while APP level and relative phosphorylation level of tau were significantly increased.In white matters of PTZ-kindled rats,reduction of NF-H and total tau,elevation of APP and relative phosphorylation level of tau were also seen.And such impairments were exacerbated as PTZ-kindling continued.Conclusions: Axonal impairment characterized by reduction of NF-H and total tau,accumulation of APP was found in brain tissues of refractory epilepsy patients and PTZ-kindled rats,accompanied with increase of relative phosphorylation level of tau.Part Two The role of NMDA receptor,GSK-3β and Cdk5 in axonal impairment of epilepsyObjectives: To explore whether NMDA receptor,GSK-3β and Cdk5 play roles in the axonal impairment of epilepsy.Methods: Rats successfully kindled with PTZ for 21 continuous days received peritoneal injection of memantine(non-selective NMDA receptor antagonist),ifenprodil(NR2B selective NMDA receptor antagonist),lithium chloride(GSK-3β antagonist),Roscovitine(Cdk5 antagonist)or saline daily for 7 days.NF-H and APP level in corpus callosum were measured using immunohistochemistry.NF-H,APP,total tau,p-Tau S396 and p-Tau T231 level in white matters of PTZ-kindled rats were also measured using western blot.And the relative phosphorylation level of tau was calculated.Results: Peritoneal injection of memantine and ifenprodil alleviated PTZ-kindling-induced NF-H reduction and APP accumulation in white matters of PTZ-kindled rats,and reversed reduction of total tau and increase of relative phosphorylation level of tau.Lithium chloride and roscovitine also alleviated PTZ-kindling-induced reduction of NF-H and increase of APP and relative phosphorylation level of tau in white matters of PTZ-kindled rats.Lithium chloride also reversed total tau reduction induced by PTZ-kindling,while roscovitine did not affect total tau level in PTZ-kindled rats.Conclusions: Inhibiting NMDA receptor,GSK-3β and Cdk5 can alleviate axonal impairment in PTZ-kindled rats,indicating that activation of NMDA receptor,GSK-3βand Cdk5 participate in epilepsy-induced axonal impairment.Part Three The molecular mechanism of NMDA receptor-mediated axonal impairment in epilepsyObjectives: To identify whether NMDA receptor mediates axonal impairment in epilepsy via activating GSK-3β and Cdk5.Methods: Rats successfully kindled with PTZ for 21 continuous days received peritoneal injection of memantine(non-selective NMDA receptor antagonist),ifenprodil(NR2B selective NMDA receptor antagonist),lithium chloride(GSK-3β antagonist),Roscovitine(Cdk5 antagonist)or saline daily for 7 days.Protein level of total GSK-3β,phosphorylated GSK-3β Y216(p-GSK-3β Y216),phosphorylated GSK-3β S9(p-GSK-3β S9)and Cdk5 in white matters of PTZ-kindled rats were measured using western blot.And m RNA level of GSK-3β and Cdk5 were measured using polymerase chain reaction(PCR).Results: Memantine and ifenprodil decreased the m RNA and protein level of Cdk5,and inhibited the activity of GSK-3β by increasing p-GSK-3β S9 and decreasing p-GSK-3β Y216.In addition,different from memantine,ifenprodil did not affect m RNA and protein level of total GSK-3β.Conclusions: NMDA receptors,especially NR2B-containing NMDA receptors,mediate axonal impairment in epilepsy via activating Cdk5 and GSK-3β.