| Objective: A model of global cerebral ischemia-reperfusion wasestablished by using a method of modified Pulsinelli’s four-vessel occlusion inSD rat, LY294002was administered to inhibit PI3K/Akt activity before globalcerebral ischemia and nasopharyngeal cooling was applied to reduce headtemperature. The purpose of this study was to observe the impact ofnasopharyngeal cooling to global cerebral ischemia-reperfusion injury,through expressions of pAkt, Akt and pFoxO3a protein in hippocampal CA1region. The effects of mild head hypothermia and the combination of headhypothermia and LY294002on global cerebral ischemia-reperfusion injurywere investigated.Methods:1.Experimental groupsSixty clean grade male SD rats weighing250~280g,aged3to4monthswhich was provided by the Experimental Animal Center of Hebei MedicalUniversity,were used in this study and randomly divided into five groups bythe Random number table(n=12): Sham group(group S); Global cerebralischemia-reperfusion injury group(group I/R); Mild hypothermia group(groupHI/R); Combination of DMSO and mild hypothermia group(HI/R+DM);Combination of LY294002and mild hypothermia group(groupHI/R+LY).2.Preparation of animal modelThe application of improved Pulsinelli four-vessel occlusion was uesed toprepare global cerebral ischemia and reperfusion model.The time of globalcerebral ischemia is15minutes and reperfusion time8hours,with roomtemperature maintained at23~25℃.All rats were anesthetized withintraperitoneal10％chloral hydrate（3.5ml/kg）. Animals are fixed at the experimental stage by prone position.The back of the neck ’s skin was cut inthe middle of the first and second cervical vertebra,to expose the double flankholes, cervicalhe bilateral vertebral arteries were electrocauterized. Aftertwenty-four hours, the trachea was cannulated via endotrachealintubation.,anesthetized with the same method.the rat was kept spontaneousbreathing. The caudalis vena of rat was inserted24G trocar. Sodium lactatedRinger’s solution was infused.the skin of Anterior midline was cut, thebilateral common carotid artery was separated,with4-0silk through thecarotid artery. Then the experimental groups were given different treatment.Ingroup S, rat received the same surgical procedures except that the bilateralvertebral arteries were not electrocauterized and the bilateral common carotidarteries were not occluded. In group I/R, global cerebral ischemia-reperfusionwas produced. In group HI/R, HI/R+DM and HI/R+LY, global cerebralischemia-reperfusion was produced same with group IR. A cotton ball and asuction device was placed in the lower pharynx to avoid aspiration ofphysiological saline. The stereotaxic apparatus was used to fix rats, withelectrode pierced in the scalp for continuous monitoring of EEG. pierced inthe subcutaneous of limbs for continuous monitoring the electrocardiogram(ECG).A temperature probe was placed to monitor hippocampal temperaturein the right hippocampal CA1region,by using Miniature cranial drill,in theright hippocampus CA1region (anterior fontanelle3.6mm, the midline rightnext to the open3mm) drilled a round hole with a diameter of about2mm.20min before global cerebral ischemia HI/R+DM group was slowly injectedDMSO5μl by using stereotaxic apparatus in the left ventricle.In HI/R+LYgroup，5μl of LY294002(LY294002is dissolved in DMSO, and theconcentration is25mmol/L) was given,Before global cerebral ischemia, two20G silicone tubes were inserted into both nasal cavities to applynasopharyngeal cooling and cold physiological saline (5℃) was infused at arate of100mL·min-1·kg-1until the hippocampal temperature was reduced to(33.0±0.5)℃. Then bilateral common carotid arteries were clamped for15min.Hippocampal target hypothermia was maintained for1h followed by rewarming spontaneously. During the experiment rectal temperature is alwaysmaintained at (37.0±0.5)℃, room temperature was maintained at23℃to25℃.3.Sample preparation and histological examination.3.1Using immunohistochemical method to determinate the expression ofpFoxO3a in Hippocampal CA1region and hematoxylin-eosin (HE) stainingand light microscopy detectionAfter 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 by physiological saline via ascending aorta, then wereperfused4％by paraformaldehyde (PFA) to fix tissues. Brain tissues werecarefully obtained. Samples were fixed with4％PFA and stored at4℃.These samples were used to observe the immunohistochemical expression ofp-FoxO3protein, and used in HE staining.3.2Using Western Blot to detect pAkt, total Akt protein in The hippocam-pusThe other six rats were anesthetized again in each group, and then werekilled by decapitation, brain tissues were quickly obtained. Samples werefrozen in liquid nitrogen, and were stored at－80℃. These samples wereused to observe the expression of pAkt protein and Akt protein.Results:1. There were no statistical differences in body weight of the rats in anyof the groups (P>0.05).2. The results of light microscope.Group S: The morphology and structure of neurons were normal in thehippocampal CA1region. The cells were neatly arranged, evenly distributed,with abundant cytoplasm, large nuclei(round or oval) and prominent nucleoli.Group I/R: The morphology and structure of neurons were severely damaged.The cell layers became little, disordered, with shrunken cells. The cell gapswere increased. Neurons were decreased in number and reduced in volume,exhibiting pyknotic nucleus and chromation condensation. Swelling cells and the cell debris could be seen. Group HI/R: Compared with group I/R,histopathological changes in the hippocampal CA1region were minor ingroup HI/R. The living cells in group HI/R were significantly greater thanthose in group I/R. The arragement of cells was slightly discordered. A smallamount of cells shrinkaged. Group HI/R+LY: Compared with group HI/R, Themorphology and structure of neurons were severely damaged. The cell layersbecame little, disordered, with shrunken cells. The cell gaps were increased.Neurons were decreased in number and reduced in volume. Swelling cells andthe cell debris could be seen.Group HI/R+DM: Compared with groupHI/R+LY, histopathological changes in the hippocampal CA1region wereminor in group HI/R+DM. The living cells in the group HI/R+DMweresignificantly greater than those in group HI/R+LY. The arragement of cellswas slightly discordered. Compared with group HI/R,there is no significantlydifference.3. The comparison of expression of pAkt protein and Akt protein inhippocampus CA1region(Western blot).Compared with group S, the expression of pAkt protein was higher ingroup I/R,group HI/R, group HI/R+DM and group HI/R+LY. Compared withgroup I/R, the expression of pAkt protein was higher in group HI/R, groupHI/R+DM and group HI/R+LY. Compared with group HI/R, the expression ofpAkt protein was significantly lower in group HI/R+LY. The differeces werestatistically significant (P<0.05).There is no statistically significant differences in the comparison ofexpression of Akt protein in hippocampus CA1region(P>0.05).4. The comparison of expression of pFoxO3a protein in hippocampusCA1region(IHS).Compared with group S, the expression of pFoxO3a protein was higherin group I/R, group HI/R, group HI/R+DM and group HI/R+LY. Comparedwith group I/R, the expression of pFoxO3a protein was higher in group HI/R,group HI/R+DM and group HI/R+LY. Compared with group HI/R, theexpression of pFoxO3a protein was significantly lower in group HI/R+LY. The differeces were statistically significant (P<0.05).5. The comparison of expression of Bcl-2(IHS)、Bax(IHS) and Bcl-2/Baxratios in hippocampus CA1region.Compared with group S, the expression of Bcl-2protein and theBcl-2/Bax ratio was higher in group HI/R, group HI/R+DM, the expression ofBax protein was higher in group I/R, group HI/R, group HI/R+DM(P<0.05);Compared with group I/R, the expression of Bcl-2protein and Bcl-2/Bax ratiowas higher, the expression Bax protein was lower in group HI/R and groupHI/R+DM(P<0.05); Compared with group HI/R and group HI/R+DM theexpression of Bax protein was higher, the expression of Bcl-2protein andBcl-2/Bax ratio was significantly lower in group HI/R+LY(P<0.05).Conclusions：1. Mild head hypothermia can alleviate global ischemia-reperfusioninjury and have neuro-protective effects in rats.2. Mechanisms of the effects of mild head hypothermia are likelyassociated with PI3K/Akt pathway, and pFoxO3a in the downstream is onepoint of the effects. |
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