Epilepsy Continuous State Of Mild Hypothermia Neuroprotective Mechanism Of Action

PART ONE:The Neuroprotective Effect of General Mild Hypothermia on nippocampus in Pilocarpine-induced Status Epilepticus inYoung RatsObjective:To investigate the ncuroprotection of mild hypothermia on hippocampal neurons in young rats with pilocarpine induced status cpilepticus(SE).The different duration of mild hypothcrmia(2h,4h,8h) were used to the rats after SE for 30mins and 60 mins.Methods:42 Male Wistar rats aged 21-days weighing 60-65g were used in this study. Animals were given pilocarpine 380mg/Kg through intraperitoneal injection to induce status epilepticus.Rats with induced-sustaining status epilepticus for 30 minutes were divided into seven groups,including:SE 30 minutes treated with NS,with diazepam 10mg/Kg,diazepam with combination of mild hypothermia for 2 hours,4 hours and 8 hours,status epilepticus for 60 minutes were treated with diazepam 10mg/Kg or diazepam with combination of mild hypothermia for 2 hours.The control group received equal volume injections of saline.72 hours after SE,the hippocampal pathological changes have been observed through HE staining Nissl staining,In order to evaluated the changes of microtubule-associated protein 2(MAP2) and caspase 3 in the rat hippocampus,immunohistochemistry was performed according to previously established methods.We also did terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling(TUNEL) to observe the PCD of hippocampus neurons.Results:The hippocampal neuronal damage were found in rats after 30 min and 60 min SE.The neurons showed pyknotic nuclei and shnmken plasma in cyton.Nissl granula decreased in CA1 and CA3 regions of hippocampus.The percentage of neuron necrosis of hippocampus were significantly more than that in control group(P＜0.05).2 hours after therapeutic alliance with diazepam and mild hypothermia, necrotic neurons in CA3 region of hippocamous were significantly reduced compared with the group treated with diazepam alone(P＜0.05).8 hours after treatment,the neuron damage in hippocampus and the percentage of necrotic cells were similar to those of the NS group.Compared with the group of diazepam treatment,the expression of MAP-2 protein were enhanced in the hippocampus with mild hypothermia with 2h,4h or 8h duration(P＜0.05).The staining intensity and immunoreactivity increased 8 h after the mild hypothermia treatment.There was no significant difference in MAP2 immunoreactivity between the 60 mins SE rats and with 2h hypothermia treatment.The expression of caspase-3 increased in the hippocampus of rats with 30 mins and 60 mins SE.Significant reductions in caspase-3 immunoreactivity were seen after the intervention of mild hypothermia.TUNEL staining showed DNA fragmentation positivity in the SE with diazepam but decreasing significant in the groups with mild hypothermia.Double stained neurons with caspase3 and NeuN indicated that the positive apoptosis neurons decreased significantly after 8 hours mild hypothermia treatment..Conclusion:1.Status epilepticus can induces brain damage such as neurons necrosis,apoptosis and the collapse of neuron cystoskeleton.2.Mild hypothermia can protect hippocampal neurons from necrosis,apoptosis and lossing of MAP-2.3.Compared with 2 and 4 hours of persistent mild hypothermia treatment,8 hours of treatment have most neuroprotection effects after SE.. PART TWO:The Mechanism of Neuroprotection with Mild Hypothermia in Pilocarpine-induced Status Epilepticus in Young RatsObjective:Study on expression of mRNA and protein of GluR1,GluR2 and metabotropic glutamate receptors mGluR1 a after SE.To explore the mechanism of neuroprotection of mild hypothermia on hippocampal neurons in young rats.Methods:A total of 50 Male Wistar rats aged 21-days were randomly assigned into 5 groups:NS group,diazepam group,diazepam with 2h,4h,and 8h mild hypothermia treatment groups.Animals were given pilocarpine 380mg/Kg through intraperitoneal injection to induce status epilepticus,72 hours after SE,the changes in GluR1 and GluR2 as well as mGluR1 a protein levels were determined using Western-blotting analysis.The protein bands were quantified by densitometric analysis.Real-time PCR was used to investigate the mRNA expression of GluR1,GluR2 and mGluR_α.Results:After pilocarpine-induced status epilepticus,the GluR1 immunopositive nrurons increased obviously in CA1 and CA3 area of hippocampus,however protein expression of GluR2 obviously reduced compared with NS group(P＜0.05).After therapeutic alliance with diazepam and mild hypothermia,GluR1 protein expression in CA1 and CA3 reduced while GluR2 protein expression increased.After 8 hours of treatment,both protein expression were significant increased compared with the group treated with diazepam alone(P＜0.05).Through Western-blotting,after 4 hours and 8 hours therapeutic alliance,the expression GluR1 and GluR2 protein in hippocampal decreased significantly compared with the group treated with diazepam alone(P＜0.05).The expression of mGluR1_αprotein increased after status epilepticus and reduced by therapeutic alliance with diazepam and mild hypothermia.With 8 hours therapeutic alliance,there was a significant reduction in mGluR1_αprotein expression compared with the group treated with diazepam alone(P＜0.05).The mRNA expression of GluR1 and mGluR1_αincreased but the expression of GluR2 mRNA decreased.With diazepam and mild hypothermia treatment,the mRNA expression of GluR1 and mGluR1_αdecreased while GluR2 increased.With 8 hours therapeutic alliance,GluR1 and mGluR1_αmRNA expression decreased significantly compared with the group treated with diazepam alone(P＜0.05) There was no significant up-regulation of GluR2 mRNA in different groups.Conclusion:GluR1,GluR2 and mGluR1_αplay an important role in the brain injury induced by SE.The protein and mRNA for GluR1,GIuR2 and mGluR1_αwere different regulated by hypothermia treatment.Hypothermia attenuated neuron injury through down-regulating the expression of GluR1 as well as mGluR1_αmRNA and protein,and up-regulating the protein expression for GluR2.