Mild Hypothermia Promotes The Expression Of GLT-1 By Up-Regulating The PI3K/Akt Pathway To Protect Against Injury Induced By Oxygen Glucose Deprivation/Reoxygenation

The phosphatidylinositol-3-kinase/protein kinase B(PI3K/Akt)signaling pathway,as an important pathway,plays an important role in ischemic stroke.Glutamate(Glu),as the main neurotransmitter in the central nervous system,is released in large quantities during cerebral ischemia,resulting in excitotoxicity and neuronal apoptosis in ischemic stroke.The up-regulation of glutamate transporter-1(GLT-1)on astrocyte membrane can promote the uptake of Glu,thus playing a neuroprotective role.In some conditions,the PI3K/Akt signaling pathway can participate in the regulation of GLT-1.Studies have shown that the mild hypothermia can activate the PI3K/Akt signaling pathway or increase the GLT-1 protein expression,reducing neuronal apoptosis and cerebral ischemia-reperfusion injury.However,it is not clear whether PI3K/Akt signaling pathway is involved in the regulation of GLT-1.Therefore,the purpose of this study was to investigate whether mild hypothermia can increase the expression of GLT-1 by up-regulating the PI3K/Akt pathway after oxygen-glucose deprivation / reoxygenation(OGD/R)injury.Part 1 The co-culture of primary neuron-astrocyte and establishment of oxygen-glucose deprivation/reoxygenation(OGD/R)injury model in SD rats.Objevtive: To establish the of primary neuron-astrocyte cocultures and oxygen glucose deprivation/reoxygenation(OGD/R)injury model.Methods:The primary neuron-astrocyte cocultures were originated from the cerebral cortex of neonatal SD rats within 24 h.The astrocytes were cultured in DMEM/F12 of 10% FBS,purified and passaged at about 7 days.After the cells of second generation covered with about 90% of the culture plates,the mature astrocytes were identified by immunofluorescence.The neuron cultures were conducted in Neurobasal+B27 serum-free medium.The neurons were identified by cell immunofluorescence when cultured for about 7-11 days.In neuron-astrocyte cocultures,the purified second-generation astrocytes that were passaged to the round glass slides were transferred into culture plates in which neurons were cultured for about 9-11 days and then cocultured with neurons in Neurobasal+B27 serum-free medium for 1-2 days.The neuron-astrocyte OGD/R injury model was established by a three-gas incubator combined with glucose-free DMEM.Oxygen glucose deprivation for 4 h and reoxygenation for 24 h.The OGD/R injury model was evaluated by optical microscopy,thionin-staining and CCK-8 kit.Results:1.The primary cortical neurons and astrocytes cultured in vitro grew in good condition with good morphology.2.The identification rate of primary astrocytes and neurons were over 95% and 90% respectively.3.In the control(Ctrl)group,the neuronal membranes were smooth and the cell structure was integrated.No edema,cavity or swelling was observed.The neuron processes were interwoven into a network structure.The neurons in the OGD/R group were obviously swollen,the cell structure was damaged,and the processes were reduced or broken.4.The number of Nissl bodies in the Ctrl group is large and dense,the dyeing is deep,and the edges are clear.The number of Nissl bodies in the OGD/R group is reduced,the dyeing is shallow,and the edges are blurred.5.The neuronal viability of the OGD/R group was significantly lower than that of the Ctrl group(P< 0.05).Conclusions:1.The primary cultured neurons and astrocytes of SD rats can meet the requirements of further experiments.2.The neuron-astrocyte OGD/R injury model was successfully established.Part 2 Mild hypothermia promotes the expression of GLT-1 by activating the PI3K/Akt pathway to protect against injury induced by oxygen glucose deprivation/reoxygenation.Objevtive: To study the relationship among the mild hypothermia(33℃)and PI3K/Akt signaling pathway and GLT-1 expression in OGD/R.Methods:According to the first part,the cocultures of neuron-astrocyte were performed.The cells cultured in vitro were randomly divided into 6 groups(n=6): Control(Ctrl)group,OGD/R group,mild hypothermia+OGD/R(HOGD/R)group,HOGD/R +LY group,HOGD/R +DHK group and HOGD/R +DM group.All but the Ctrl group was exposed to OGD for 4h,followed by reoxygenation for 24 h,and the mild hypothermia duration is consistent with the time of the OGD/R injury.The neuronal viability in each group was compared by optical microscopy,thiopurine staining and CCK-8 kit.The expression of p-Akt and GLT-1 was detected by immunofluorescence and Western Blotting.The concentration of extracellular glutamate was measured by glutamic acid assay kit.Results:1.Compared with Ctrl group,the neuronal viability of other five groups was significantly decreased(P<0.05).Compared with HOGD/R group,the neuronal viability of OGD/R group,HOGD/R+LY group and HOGD/R +DHK group significantly decreased(P<0.05).2.Compared with Ctrl group,the intracellular green fluorescence of OGD/R group was very weak and the expression of p-Akt protein was significantly decreased(P<0.05).Compared with HOGD/R group,the intracellular green fluorescence of OGD/R group and HOGD/R +LY group was weak,and the expression of p-Akt protein was significantly decreased(P<0.05).3.Compared with Ctrl group,the intracellular red fluorescence of OGD/R group was very weak and the expression of GLT-1 protein was significantly decreased(P<0.05).Compared with HOGD/R group,the intracellular red fluorescence of OGD/R group and HOGD/R +LY group was weak,and the expression of GLT-1 protein was significantly decreased(P<0.05).4.Compared with Ctrl group,the concentration of Glu in other groups was significantly higher than that in Ctrl group(P<0.05).Compared with HOGD/R group,the concentration of Glu in OGD/R group,HOGD/R +LY group and HOGD/R +DHK group was higher than that in HOGD/R group(P<0.05).Conclusions:1.Mild hypothermia can reduce the neuronal damage after oxygen glucose deprivation / reoxygenation in neuron-astrocyte cocultures.2.The protective mechanism of mild hypothermia may be related to the increased expression of GLT-1 protein by upregulating the activity of PI3K/Akt signaling pathway,reducing extracellular glutamate concentration.