Effect Of Focal Mild Hypothermia On Expression Of PTEN After Global Cerebral Ischemia And Reperfusion In Rats

Objective: A rat head mild hypothermia was established by using amethod of nasopharyngeal cavity cooling. Pulsinelli's four-vessel occlusionwas used to produce global cerebral ischemia and reperfusion. The effect oflocal mild hypothermia on expression of PTEN at different time point afterreperfusion was observed, and the MDA content, SOD activity, S100B proteincontent in rat brain was measured, and the expression of Bcl-2protein, Baxprotein in hippocampal CA1region was observed. The mechanism of mildhypothermia neuroprotection against cerebral ischemia and reperfusion injurywas investigated.Methods:1. Experimental groups and preparation of animal model1.1Experimental groupSixty-six healthy adult male Wistar rats weighing230-280g (from theExperimental Animal Center of Hebei Medical University) were selected. Therats randomly divided into3groups: sham group (group S), normothermiacerebral ischemia and reperfusion group (group I/R), hypothermia cerebralischemia and reperfusion group (group HI/R). Among them, group I/R andgroup HI/R each randomly divided into5subgroups according to differentreperfusion time (the reperfusion time was4h,8h,12h,24h,72h),there weretotal11groups, n=6.1.2Preparation of animal model and experiment procedureAnaesthesia was induced by intraperitoneal inject10%chloral-hydrate.The skin of the middle back of the neck was incised, the muscle layer by layerwas separated, and vertebral arteries in the right and left pterygoid hole wereburn to block permanently by using60watts electric iron in group I/R andgroup HI/R. Rats in sham group were only exposed pterygoid hole without vertebral arteries blocked. Twenty-four hours later, the rat was anesthetizedagain, and trachea was cannulated via endotracheal intubation, oxygen wasinhaled and spontaneous breathing was retained. Needle electrodes wereinserted into rat's bilateral top or limbs, and electroencephalogram (EEG) andelectrocardiogram (ECG) was monitored continuously. Rat's tail vein wascatheterized for infusion of Ringer lactate liquid at a speed of1ml/h. Rat'scarotid arteries of group I/R was clamped at this step. In group HI/R, Asilicone tube was inserted into rat's nasal cavity and fixed. Rat of group HI/Rwas placed in a stereotaxic apparatus, it's scalp was cut and periosteum wasexposed. Rat's skull was drilled broken using a micro-cranial drill afterfinding hippocampus surface signs. The temperature probe was placed intohippocampal CA1section under periosteum2mm.4℃cold saline was infusedto rat's nasopharyngeal cavity until the hippocampal temperature decreased to33±0.5℃, and hypotermia was maintained for1h in group HI/R. Rat'scarotid arteries in group HI/R were clamped when it's brain temperature reachto33℃. Rat's carotid arteries in group sham were only exposed but notclamped. Rat's neck in group I/R and group HI/R was cut to make alongitudinal incision, then separated muscle and exposed carotid arteries, passthrough common carotid arteries using surgical thread and clamp them usingartery-clip, the blocked time was15min. In experiment, chloral hydrate wasgiven intermittent according to depth of rat's anesthesia.2. Specimen collection and testing methods2.1Specimen collectionThe rat was anesthetized and decapitated according to different reperfusiontime. Rat's cranial cavity opened, and whole brain was removed. The lefthemisphere was dipped into4%paraformaldehyde solution, and used forimmunohistochemistry to observe the expression of P-PTEN, Bcl-2, BaxProtein.The right hemishere was put into liquid nitrogen, and then stored inRefrigerate that-70℃for detecting the MDA content, SOD activity andS100B Content.2.2Testing methods 2.2.1The testing methods of MDA,SOD,S100BRight brain tissue was mixed with0.9%saline to made10%and1%tissuehomogenates. Tissue homogenates were centrifuged using centrifuge at4000rev/min low temperature for10min, take supernatant. Among them,1%tissue homogenate detected SOD activity. SOD activity detected by xanthineoxidase.10%tissue homogenate detected content of MDA and S100B. MDAwas detected by thiobarbituric (i.e. TBA method). S100B protein was detectedby double antibody sandwich enzyme-linked immunosorbent assay (ELISA).2.2.2The testing methods of P-PTEN,Bcl-2,BaxThe left hemisphere brain was removed and cut to5mm brain tissue. Braintissue that trim well were dehydrated, transparent,dip wax,embedded,sectioned,dewaxed,into the water,antigen retrieval,closed and othermeasures conventionally. Detection of Bcl-2, bax, P-PTEN use SP metherd.The detected steps accorded for the requirements of the appropriate kit instrict.Results：1. Comparison of P-PTEN content in each groupsCompared with group sham, content of P-PTEN of group I/R decreasedat reperfusion4h,8h,12h,24h,72h, There had statistically significanceBetween two groups,(P <0.05). Compared with group I/R, content of P-PTENOf group HI/R increased at same reperfusion time. There had statisticallySignificance between two groups,(P <0.05). In group I/R, content of P-PTENBegan to decrease at reperfusion8h, it significantly reduced at reperfusion12h,Decreased the minimum value at reperfusion24h, and content of P-PTENElevated at reperfusion72h but still lower than that of group sham.2Comparison of Bcl-2, Bax in each groupsCompared with group sham, Bcl-2decreased and Bax increased in groupI/R, There had statistically significance between two groups (P <0.05).Compared with group I/R, Bcl-2of group HI/R increased at reperfusion12h24h, Bax of group HI/R decreased at reperfusion8h,12h,24h,72h,There hadStatistically significance between two groups,(P <0.05). In group I/R, Expression of Bcl-2began to decrease at reperfusion8h, it decreased at theLowest value at reperfusion24h then increased gradually. Expression of BaxIncreased gradually following extend of reperfusion time.3Comparison of MDA, SOD in each groupsCompared with group sham, content of MDA of group I/R increased,activity of SOD of group I/R decreased. There had statistically significancebetween two groups (P <0.05). Compared with group I/R, content of MDAof group HI/R decreased. Activity of SOD of group HI/R increased. There hadstatistically significance between two groups (P <0.05). In group I/R,content of MDA increased gradually with the extension of reperfusion time.They reached the highest value at reperfusion24h then reduced gradually butstill higher than that of group sham. Activity of SOD decreased gradually withthe extension of reperfusion time. They reached the lowest value atreperfusion24h then increased gradually.4Comparison of S100B level in each groupsCompared with group sham, the level of S100B of group I/R increased,There had statistically significance between two groups (P <0.05). Comparedwith group I/R, the level of S100B of group HI/R decreased at samereperfusion time, There had statistically significance between two groups,(P<0.05).Conclusion:Expression of p-PTEN decreases in hippocampal CA1region aftercerebral ischemia and reperfusion injury. Hypothermia could increaseexpression of p-PTEN in the hippocampal CA1region after cerebralischemia-reperfusion. Mild hypothermia reduces cerebral ischemia andreperfusion injury through inhibiting the activity of PTEN and apoptosis, andreduceing production of oxygen free radical.