Effects Of Mild Hypothermia On The Expression Of Glutamate Transporter And Apoptosis In Rats With Cerebral Ischemia Reperfusion Injury

Objective: To investigate the effects of mild hypothermia on the expression of GLT-1 and apoptosis in rats global cerebral ischemia reperfusion injury,explore the neuroprotective mechanism of mild hypothermia and provide references for the application of hypothermia therapy in clinical.Methods:258 healthy male Wistar rats(250~300 g)were randomly divided into seven groups by random number method.1)Control group(n=42): Decapitated directly without any operation.2)Sham group(n=42): Rats in the sham groups were only exposed bilateral vertebral artery and carotid artery without occluding blood flow.3)Group VOA(n=42): Blocked the bilateral vertebral arteries and exposed but not occluded the bilateral common carotid arteries 48 hours later.4)Group NI/R(n=42): The rats were subjected to global brain ischemia for 8 min,maintaining the temperature of hippocampal and rectal at 37 ± 0.5 ? for 2 h.5)Group HI/R(n=84): The rats were subjected to global cerebral ischemia for 8 min maintaining the head temperature at 33 ± 0.5 ? for 2 h while the rectal temperature at 37±0.5?,and than rewarmed spontaneously.6)Group NS+HI/R(n=42): The rats were administered normal saline 30 min before global cerebral ischemia for 8 min and the temperature of hippocampal was maintained at 33 ± 0.5 befor? e the onset of ischemia while the rectal temperature was maintained at 37 ± 0.5 ?for 2 h,and than rewarming spontaneously.7)Group DHK+HI/R(n=42): The rats were administered DHK 30 min before global cerebral ischemia for 8 min,the head temperature was maintained at 33 ± 0.5 ?for 2h while the rectal temperature was maintained at 37 ± 0.5?,and than rewarming spontaneously.Rats in control group were decapitated directly without any treatment.The remaining rats in each group were killed by decapitation at predetermined time points(n=6).Rat brains in group 1~5 were incised follow the brain midline.The left side of the brain tissue fixed with 4 % paraformaldehyde and be made to 5 ?m paraffin sections to observe the pathological changes by thionine staining and the expression of GLT-1,Bax and Bcl-2 protein by immunohistochemistry in the hippocampus.Rapidly separated the CA1 region of the right side of the brain tissue,frozen in liquid nitrogen and stored in-80 ? refrgerator for western blot analysis of the expression of GLT-1 protein.Group 6 and 7 were decapitated and fixed with 4% paraformaldehyde for thionine staining to observe the pathological changes.Results: 1 The pathology results of light microscopy(thionine staining).Pyramidal neurons in the hippocampal CA1 area in Group control were arranged in order,the HG was 0 and ND was 214±3.7.There was no obvious neuronal injury in sham group at every time points.HG and ND have no significant differences compared with control group(P>0.05).At the 7 days,almost all neurons died after cerebral ischemia reperfusion in NI/R group,HG increased to ?grade and ND(42±3.4)decreased significantly.While only sporadic nucleus pyknosised in group HI/R,compared with NI/R group rats at the seventh days,HG(0 ~?)decreased obviously(P< 0.05)and ND(187 ± 3.0)increased significantly(P< 0.05).No obvious neuronal damage was found at the seventh days after cerebral ischemia reperfusion in group NS+HI/R,while rats in group DHK+HI/R showed obvious neuronal death,HG decreased to ?~ grade? and ND was 63±2.6 having significant differences with group NS+HI/R(P< 0.05).2 Comparison of GLT-1 protein levels in hippocampus.2.1 Western blot analysisIn control group,the expression level of GLT-1 has always been a basic level.GLT-1 in sham group showed no significant differences with the group control(P>0.05).In group VOA,GLT-1 began to express at 0 h,peaked at 8 h,decreased at 16 h,the fewest amount expression appeared at the first day and increased again at the fifth days.The expression of GLT-1 at different time points have significant differences versus the sham group(P<0.05).In group NI/R,it was found that GLT-1significantly increased to the peak at 0 h,than decreased at 8 h,decreased most significantly at the fifth days,and reached the level before ischemia at the seventh days.The expression of GLT-1 at all time points except the first day was lower than that in group VOA(P<0.05).The GLT-1 expression level in group HI/R began to increase at 0 h,peaked at 8 h,decreased at 16 h,and expressed the least mount at the fifth days,while still kept a high level of expression.The expression level of GLT-1 was higher than that in group NI/R at each time point(P< 0.05).2.2 GLT-1 immunohistochemistryGLT-1 immunoreactive particles mainly distributed in astrocytes of hippocampal CA1 region.We observed some GLT-1 immunoreactive particles at each time point in both sham group and the control group.In group VOA,GLT-1 largely expressed at 0 h,peaked at 8 h,decreased 16 h,increased again at the third day,then decreased gradually,and GLT-1 expression in each subgroup were higher than that in the group sham(P<0.05).In group NI/R,GLT-1 increased at 0 h,peaked at 8 h,decreased significantly at 16 h~7 d.The expression of each time point were lower than those in the VOA group(P<0.05).The expression of GLT-1 in group HI/Rat each time point except 0 h were higher than that in group NI/R(P<0.05).3 Comparison of Bcl-2 and Bax expression in hippocampal 3.1 Bax immunohistochemistryThe immunoreactive markers of Bax presented brown granular deposition mainly distributed in both cytoplasm and synapses.There was little Bax expression at different time points in control group and sham group rats,and had no differences with each other(P>0.05).Rats in group VOA started a little Bax expression at 0 h,peaked at 16 h,than decreased gradually.Bax at each time point was slightly higher than that in sham group(P< 0.05).In NI/R group,Bax began to express largely at 0 h,peaked at the third days and reduced obviously at the fifth and seventh days,and Bax expression at all time points was significantly higher than that in group VOA(P< 0.05)except the fifth and seventh days.In group HI/R,Bax started a small amount of expression at 0 h,peaked at 16 h,than decreased,but the expression levels at each time point still significantly lower than that in NI/R group(P < 0.05).3.2 Bcl-2 immunohistochemistryThe immunoreactive markers of Bcl-2 presented brown granular deposition located both cytoplasm and the start of synapses.The control group and sham group rats only expressed a little at each time point.Group sham and group control had no significant differences with each other(P>0.05).Bcl-2 in group VOA began to express at 0 h,peaked at 1~3 days,than decreased gradually.Bcl-2 in group NI/R began to express at 0 h,peaked at the first day,reduced obviously at the third day,declined to the levels before ischemia at the seventh days and the expression levels of Bcl-2 in every time points were lower than that in VOA group(P<0.05).Bcl-2 in group HI/R increased at 0 h,peaked at 8 h,than decreased slightly and still kept a high level expression at 7 days.The Bcl-2 at each time point in group HI/R were significantly higher compared with group NI/R(P<0.05).Conclusion:1 Head mild hypothermia can reduce the number of necrosis of neuron in CA1 after ischemia reperfusionon and has a neuroprotection.2 Head mild hypothermia can inhibit the down-regulation of GLT-1after cerebral ischemia reperfusion injury.3 GLT-1 may be involved in neuroprotection of head mild hypothermia.