| Objective: A model of global cerebral ischemia-reperfusion wasestablished by using a method of modified Pulsinelli's four-vessel occlusion inrat, bpv(pic) was administered to inhibit PTEN activity before global cerebralischemia and nasopharyngeal cooling was applied to reduce head temperaturein rats. The purpose of study was to detect the expression of P-PTEN protein,Bax protein, Bcl-2protein in hippocampal CA1region, to determineconcentration of S100B protein, MDA content and SOD activity in braintissue. The effects of inhibition of PTEN activity, mild head hypothermia andtheir combination on global cerebral ischemia-reperfusion injury in rats wereinvestigated.Methods: Sixty healthy male Wistar rats weighing250~280g were usedin this study and randomly divided into five groups(n=12each): Shamoperation group(group S); Global cerebral ischemia-reperfusion injurygroup(group IR); Inhibition of PTEN activity group(group B); Mildhypothermia group(group MH); Combination of inhibition of PTEN activityand mild hypothermia group(group MB).All rats were anaesthetized with intraperitoneal10％chloral hydrate(350mg/kg). The bilateral vertebral arteries were electrocauterized. Aftertwenty-four hours, the trachea was cannulated via endotracheal intubation.The rats kept spontaneous breathing. The vena caudalis of rat was cannulatedwith24G trocar. Sodium lactated Ringer's solution was infused. After rat wasplaced in a stereotaxic apparatus, electroencephalogram (EEG),electrocardiogram (ECG) and the rectal temperature were continuouslymonitored. Rectal temperature was continuously maintained at (37.0±0.5)℃by changing a hot water bag under the truncus of a rat and regulating the height of an incandescent lamp up the truncus of a rat. The bilateral commoncarotid arteries were blocked for15min followed by8h reperfusion. we useddifferent methods according to different groups. In group S, rats received thesame surgical procedures except that the bilateral vertebral arteries were notelectrocauterized and the bilateral common carotid arteries were not occluded.In group B, global cerebral ischemia-reperfusion was produced, rats weregiven bpv(pic) i.p. to inhibit PTEN activity at a dose of20μg/100g for fourtimes with an interval of3h and ischemia was induced after the last injection.In group IR, global cerebral ischemia-reperfusion was produced, rats weregiven an equal volume physiological saline by intraperitoneal injectes. Ingroup MH and MB, global cerebral ischemia-reperfusion was produced. Acotton ball and a suction device were placed in the lower pharynx to avoidaspiration of physiological saline. A temperature probe was placed to monitorhippocampal temperature in the right hippocampal CA1region. Before globalcerebral ischemia, two20G silicone tubes were inserted into both nasalcavities to apply nasopharyngeal cooling and cold physiological saline (5℃)was infused at a rate of100mL·min-1·kg-1until the hippocampal temperaturewas reduced to (33.0±0.5)℃. Then bilateral common carotid arteries wereclamped for15min. Hippocampal temperature was maintained for1hfollowed by rewarming spontaneously. In group MB, rats were given bpv(pic)by intraperitoneal injectes and nasopharyngeal cooling was applied beforeglobal cerebral ischemia. In group MH, except that nasopharyngeal coolingwas applied, rats were given an equal volume physiological saline byintraperitoneal injectes before global cerebral ischemia.After rats were observed for8h in group S and reperfused for8h in othergroups, six rats selected randomly were anesthetized again in each group. Ratswere firstly perfused physiological saline via ascending aorta, then perfused4％paraformaldehyde (PFA) to fix tissues. Brain tissues in rats were carefullyobtained. Samples were fixed with4％PFA and stored at4℃. These sampleswere used in immunohistochemical test to observe the expression of P-PTENprotein, Bcl-2protein and Bax protein. The another six rats were anesthetized again in each group. Then theywere killed by decapitation, brain tissues were quickly obtained. Samples werefrozen in liquid nitrogen, afterwards, they were stored at-70℃. These sampleswere used to determine the concentration of S100B protein, MDA content andSOD activity.Results:1There were no statistical differences in body weight of the rats among thefive groups (P>0.05).2The comparison of expression of P-PTEN protein in hippocampus CA1region(IHS)Compared with group S, the expression of P-PTEN protein was less ingroup IR, the expression of P-PTEN protein was higher in group MB.Compared with group IR, the expression of P-PTEN protein was higher ingroup B, group MH and group MB. Compared with group B or group MH, theexpression of P-PTEN protein was significantly higher in group MB. Thediffereces were statistically significant (P<0.05).3The comparison of expression of Bax protein(IHS), Bcl-2protein(IHS) andBcl-2/Bax ratios in hippocampus CA1regionCompared with group S, the expression of Bax protein was higher inother four groups, the expression of Bcl-2protein was higher in group B,group MH and group MB, Bcl-2/Bax ratio was lower in group IR and washigher in group MH and group MB. Compared with group IR, the expressionBax protein was less in group B, group MH and group MB, the expression ofBcl-2protein and Bcl-2/Bax ratio were higher in group B, group MH andgroup MB. Compared with group B, the expression of Bax protein was less ingroup MB, the expression of Bcl-2protein and Bcl-2/Bax ratio were higher ingroup MH and group MB. Compared with group MH, the expression of Baxprotein was less, the expression of Bcl-2protein and Bcl-2/Bax ratio werehigher in group MB. The differeces were statistically significant (P<0.05).4The comparison of concentration of S100B proteinCompared with group S, the concentration of S100B protein was higher in group IR, group B and group MH. Compared with group IR, the concentrationof S100B protein was less in group B, group MH and group MB. Comparedwith group B or group MH, the concentration of S100B protein wassignificantly much less in group MB. The differeces were statisticallysignificant (P<0.05).5The comparison of content of MDA and SOD activityCompared with group S, the content of MDA was higher in group IR andgroup MH, the SOD activitity was less in other four groups. Compared withgroup IR, the content of MDA was less in group B, group MH and group MB.the SOD activitity was higher in group MH and group MB. Compared withgroup B, the content of MDA was less in group MB, the SOD activitity washigher in group MH and group MB. Compared with group MH, the content ofMDA was less and the SOD activitity was higher in group MB. The differeceswere statistically significant (P<0.05).Conclusions：1. Inhibition of PTEN activity, mild hypothermia and their combination all canalleviate global ischemia-reperfusion injury in rats and all haveneuroprotective effect.2. Combination of inhibition of PTEN activity and mild head hypothermia hasmore enhanced neuroprotective effect than either used alone.3.Mechanisms of the effects are likely associated with inhibition of apoptosisand clearing of reactive oxygen species. |
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