| Objective: Blocking bilateral vertebral artery and common carotid artery caused global cerebral ischemia-reperfusion injury. Nasopharyngeal cavity cooling method was used to reduce deep brain temperature. Observational the change of hippocampal cells mitochondrial transmembrane potential during the period of ischemia-reperfusion in rats, and the hippocampal CA1 area of Cytochrome C(Cytochrome C, Cyt C) protein, the Bcl- 2 protein,Bax protein and Caspase-3 protein expression. Researched on the relation between mitochondrial membrane potential change and cell apoptosis, and the head mild hypothermia of protection mechanism of action of ischemia reperfusion brain injury. Providing theoretical basis for clinical treatment.Methods: 1 Experimental groupsThirty-six healthy Wistar rats was randomly divided into three groups, all of them were male and weighing 250~280g, there were twelve rats in each group. Sham group was marked as group S; Global cerebral ischemia-reperfusion injury group was marked as group I/R; Mild hypothermia on global cerebral ischemia-reperfusion injury group was marked as group HI/R. 2 Experimental method and the implementation of head mild hypothermiaBlocking bilateral vertebral artery and common carotid artery caused global cerebral ischemia-reperfusion injury. Nasopharyngeal cavity cooling method was used to reduce deep brain temperature. Select 8hours fasting rats, weighing after intraperitoneal injection of 10% chloral hydrate anesthesia. After the success of the anesthesia fixed the rat in work station, under the skull to touch the first cervical vertebra. Along the center line separated muscle tissue step by step until the spine. Exposed the double flank holes. Then with the electric soldering iron to cauterize the bilateral vertebral arteries to occlusion. Suture the skin. After 24 h with same method to anesthesia animal again, endotracheal intubation. Continuous sevoflurane inhalation, keep spontaneous breathing oxygen. On the center of the neck,separated the bilateral common carotid arteries, prepare artery clamp. Group S of rats only exposed to double flank holes, common carotid artery, no other processing. Group I/R, group HI/R are burned flank holes to occlude vertebral artery, and clip the bilateral common carotid arteries caused global cerebral ischemia by 15 min to establish experiment model. Nasopharyngeal cavity cooling method was used for group HI/R: Bilateral nasal cavity after endotracheal intubation in the silicone tube, continuous pumping of 4℃ cold saline cause head temperature decrease, until the temperature of hippocampus area dropped to 33.0℃, clip bilateral common carotid artery. Blood reperfusion an hour later the natural recovery temperature. 3 Sample preparation and detection3.1 Detect hippocampus mitochondrial membrane potential changes by JC- 1 fluorescent staining24 hours after reperfusion in rats in each group, after intraperitoneal injection of 10% chloral hydrate anesthesia remove the brain tissue rapidly, the separation of hippocampus on ice tray, according to the extraction kit instructions to extract mitochondria, used for observation of the mitochondrial membrane potential. The whole operation process was conducted in the low temperature environment. 3.2 Using Western Blot to detect Caspase-3 proenzyme4 rats were selected in each group. Inject 10% chloral hydrate to anesthesia the rat and remove the brain tissue rapidly, the hippocampus was frozen by liquid nitrogen at low temperature quickly and moved into the refrigerator, stored at-80℃. Used Western Blot method to detect Caspase-3 proenzyme.3.3 Detect the expressions of Cyt C protein, Bax protein,and Bcl-2 protein in hippocampal CA1 region by immunohistochemical methodAfter successful preparation of ischemia reperfusion model,with 10% chloral hydrate anesthesia in rats, with 0.9% sodium chloride infusion to clean blood vessels, then with 4% paraformaldehyde(PFA) to fix tissues. Remove the brain tissue rapidly, The samples were placed in refrigerator at 4℃ to store, Used immunohistochemical staining to detect the expressions of Cyt C protein, Bax protein,and Bcl-2 protein.Results:1 Caspase-3 proenzyme expression in hippocampal CA1 area of the ratsCompared with group S, group I/R of Caspase-3 proenzyme expression decreased, group HI/R expression decreased(P<0.05); Compared with the group I/R, group HI/R of Caspase-3 proenzyme expression increased(P<0.05).2 Cyt C protein expression in hippocampal CA1 area of the ratsCompared with group S, Cyt C protein expression were increased of group I/R and group HI/R(P<0.05); Compared with group I/R, the expression of Cyt C protein was decreased in group HI/R. The difference were statistically significant between two groups(P<0.05).3 Bcl-2 protein and Bax protein expression in hippocampal CA1 area of the ratsCompared with group S, Bcl-2 protein and Bax protein expression were increased of group I/R and group HI/R(P<0.05). Compared with group I/R, the expression of Bax protein of group HI/R was decreased, and the Bcl-2 protein was increased. The difference were statistically significant between two groups(P<0.05).4 The observation of mitochondrial membrane potential in hippocampal CA1 area of the ratsGroup S in JC-1 fluorescent staining expressed as bright red and bright green(green++red++), this is the mitochondrial transmembrane potential of normal image; Group I/R in JC-1 fluorescent staining expressed as less bright red and normal bright green(green++red),this means that the mitochondrial transmembrane potential decline obviously; Group HI/R in JC-1 fluorescent staining expressed as red and bright green(green++red+), this means that the mitochondrial transmembrane potential decline; Compared with group I/R, the contrast of group HI/R in JC-1 fluorescent staining is not obvious, this means that the decline of mitochondrial transmembrane potential of group HI/R is less than group I/R.Conclusions:1 Mild head hypothermia affect on global cerebral ischemia-reperfusion injury in rats. The protection mechanism is related to hippocampus mitochondrial membrane potential and cell apoptosis; The change of mitochondrial membrane potential is closely related to cell apoptosis.2 Mild head hypothermia can defense the global cerebral ischemia-reperfusion injury in rats and has the nervous system protective effect. |
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