| Objective:This experiment has used the method of modified Pulsinelli’s four-vessel occlusion in Wistar rats to establish a model of global cerebral ischemia-reperfusion. Before global cerebral ischemia whitch was combined with the method of nasopharyngeal cooling to implement the head temperature, Rapamycin was administered to inhibit the activity of m TOR. The purpose of this study was to detect the expressions of m TOR protein and p-m TOR protein in brain tissue, to test the expression of P70S6 K protein and p-P70S6 K protein in hippocampal CA1 region. The effects of mild head hypothermia and the combination of inhibition of m TOR signaling pathway and mild head hypothermia on global cerebral ischemia-reperfusion injury in rats were investigated, the possible mechanism of cerebral protection with hypothermia was further discussed. And this study can provide new basis for clinical prevention and treatment of cerebral ischemia.Methods: 1 Experimental groupsSixty clean grade healthy male Wistar rats weighting 250~300g, whitch were provided by the Experimental Animal Center of Hebei Medical University were used in this study and randomly divided into five groups(n=12): Sham group(group S), Global cerebral ischemia-reperfusion injury group(group I), Mild hypothermia group(group T), Combination of Rapamycin and mild hypothermia group(group R), Combination of DMSO and mild hypothermia group(group D). 2 Preparation of global cerebral ischemia-reperfusion modelThe method of modified pulsineli’s four-vessel occlusion applied to this study to prepare global cerebral ischemia and reperfusion model. All rats were weighed up and anesthetized with 10% chloral hydrate(400mg/kg) after 8 hours fasting but water free, then these rats were fixed at the animal experimental stage by prone position to be polled and sterilized. The skin was sliced in the center of the neck from the first to the second cervical vertebra to separate organization and then made the double flank holes exposed. The homemade equipment was used burning the bilateral vertebral arteries to make them inaccessible. After those processes, the skin was sutured and disinfected. After 24 hours, the rats were anesthetized again by the same method and fixed on the experimental table with supine position, then tracheal intubations were given and spontaneous respirations were retained. During the experiment, oxygen and sevoflurane were discontinuously given to the rats to maintain anesthesia. The tail vein was inserted with trocar to meet the needs of the loop by pumping lactated Ringer’s solution at the rate of 100 m L·h-1. The skin of anterior midline was polled, sterilized and sliced so that it was easy to separate organization to make the bilateral common carotid artery exposed, with 4-0 silk through the carotid artery. 3 Experimental treatments and monitoringRoom temperature was maintained at(37.0±0.5) from beginning to end ℃by putting a hot water bag under the trunk of a rat and regulating the height of an incandescent light bulb over the rat’s body. The time of global cerebral ischemia is 15 minutes and reperfusion time is 2 hours. Different group received different treatment: In group S, rats received the same surgical procedures to make the double flank holes exposed but the bilateral vertebral common carotid arteries were not implosive and to make the bilateral common carotid arteries separated but not occluded. In the other four groups, the bilateral vertebral common carotid arteries were occluded to cause global cerebral ischemia. Group T, group R and group D were hypothermal groups: Before global cerebral ischemia, tracheal intubation was given, a cotton ball and a suction device was placed in the pharynx to avoid aspiration. Tow 20 G silicone tubes were put into bilateral nasal cavities, the depth was 20 mm. Cold physiological saline(4-5℃) was infused at a rate of 100 m L·min-1·kg-1 so that the temperature of the head could be decreased. In the process of lowing temperature, an suction apparatus was used to prevent aspiration. When the temperature of hippocampus was reduced to(33.0±0.5) ℃, the bilateral common carotid arteries were clipped for 15 minutes. The low temperature was lasted for 1 hour and then rewarmed naturally. Group R and group D were medicated groups. 1h before global cerebral ischemia, a stereotaxic instrument was used to slowly inject 5μl Rapamycin(10mmol/l) into a left ventricle of rats of group R and the same volume of DMSO was given to group D. The electrode was used to pierce the scalp to supervise for EEG,to pierce the skin of limbs to monitor ECG. A temperature probe was placed in the right hippocampal CA1 region(anterior fontanelle 3.6 mm, the midline right side 3 mm and suncortical 2 mm) to monitor the temperature of hippocampus. The rectal temperature was supervised all through the test. 4 Sample preparation and detection 4.1 Immunohistochemical method was used to measure the expression of P70S6 K protein, p- P70S6 K protein in brain tissues and to determinate the hematoxylin- eosin(HE) staining and light microscopy detection.After observed for 2 hours in group S and reperfused for 2 hours in other four groups, six rats were randomly chose to be anesthetized. Then animals were perfused by 4% paraformaldehyde(PFA) to fix tissues, after this procedure, the brain tissues were fleetly got out and put in 4% PFA for fixation. Samples were kept in fridge at 4 for next determination.℃ 4.2 The method of Western Blot was used to determine m TOR protein, p- m TOR protein in the hippocampus.The other six rats in each group were anesthetized by 10% chloral hydrate(400mg/kg) and then killed with brains tissues were quickly taken out. The hippocampal tissues were carefully separated and put into liquid nitrogen to preserve and then stored at-80℃. These samples were used to monitor the expression of m TOR protein and p- m TOR protein..Results:1 The body weight of the rats in any of the groups has no statistical difference(P>0.05).2 The results of light microscope.Group S: In the hippocampal CA1 region the morphology and structure of plasma and appeared red, cell nucleus were big and were roundness or oval and they showed clear boundary. All cells were neatly arranged and homogenous distributed, and there were no abnormal morphology cells. Group I: The morphology and structure of neurons were severely damaged and normal neurons markedly reduced. Some of the cells’ body were shrinked and swelled in the cytoplasm. The shape of the cells was irregularity. A group of the cells were arranged in a mess and uneven distributed. The quantity of survival cells was decreased. Group T: Compared with group I, the degree of injury of neurous’ in the hippocampal CA1 region was lighten. A small amount of cells’ body was shrinked and swelled in the cytoplasm. The cells were arranged slightly mussily. The levels of the cells were increased. The number of survival cells was increased signally. Group R: Compared with group T, the degree of injury of neurons in the hippocampal CA1 region was memorably anabatic. The body of the cells was shrinked. And apoptotic morphological characteristics obviously appeared, such as pyknosis, karyorrhexis. The cells’ shape was not regular and arranged unstuck, and the survival cells were much less. Group D: Compared with group R, the degree of injury of neurons in the hippocampal CA1 region was palliative. The cells were arranged orderly and survival cells were escalatory. Compared with group T, there was no prominent discrepancy between the tow groups.3 The comparison of P70S6 K protein and p-P70S6 K protein in brain tissues.Compared with group S, the expression of p- P70S6 K protein was higher in group I, group T, group R and group D, and the differences were statistically significant(P<0.05). Compared with group I, the expression of p- P70S6 K protein were increased and the differences had statistical sense(P<0.05). Compared with group T, the p- P70S6 K protein in brain tissues were decreased with statistical significance(P<0.05).Compared with group R, the p- P70S6 K protein were much more in group D and the difference was statistically significant(P<0.05). There was no statistically significance among other groups(P>0.05).4 The comparison of m TOR protein and p- m TOR protein in the hippocampus.Compared with group S, the expression of p- m TOR protein were much more in other groups with statistical significance(P<0.05). Compared with group I, the expression of p- m TOR protein were increased in other groups and the differerces had statistical significance(P<0.05). Compared with group T, the expression of p- m TOR protein decreased in group D and the difference had statistical sense(P<0.05). There was no statistically significance in other groups(P>0.05).Conclusions:Mild hypothermia on a rat’s head can reverse the injury of global ischemia-reperfusion and protect the neurons.There are correlations between mechanisms of the protective effect of mild head hypothermia and the activity of m TOR signaling pathway. |
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