| Objective: Modified Pulsinelli’s four-vessel occlusion was used toestablish global cerebral ischemia-reperfusion injury in rats. To observechanges in hippocampal CA1neurons and detect the expressions of pAkt, Akt,pFoxO3a, Bcl-2and Bax protein in hippocampal CA1region, nasopharyngealcooling was applied to reduce head temperature and IGF-1was administeredvia tail vein to activate PI3K/Akt activity. The purpose of this study was todiscuss the effects of combination of mild hypothermia and IGF-1on PI3K/Aktsignaling pathway following global cerebral ischemia-reperfusion injury inrats, and to provide new basis for clinical prevention and treatment of cerebralischemia.Methods: Sixty healthy male SD rats weighing250~280g were used inthis study and randomly divided into five groups (n=12): Sham group(groupS); Global cerebral ischemia-reperfusion injury group(group C); Mildhypothermia group(group H); IGF-1group(group I); Combination of IGF-1and mild hypothermia group(group HI).Modified Pulsinelli’s four-vessel occlusion was used to establish globalcerebral ischemia-reperfusion injury in rats. All rats were anaesthetized with10％chloral hydrate（350mg/kg）via intraperitoneal injection after8hourfasting. Rats was fixed on the laboratory bench in prone position.The bilateralvertebral arteries were cauterized to occlusion. After twenty-four hours,Tracheal intubation was given after anesthesia and spontaneous respirationwas retained.Meanwhile,let the rats inhale oxygen and0.5%-1%sevoflurane.The tail vein was inserted24G trocar and Sodium lactated Ringer’s solutionwas continuous pumped at the rate of1ml/h.Bilateral common carotid wasthreaded by a silk after isolated. Then, electrocardiogram, electroence- phalogram and rectal temperature were monitored. Rectal temperature wasmaintained at37.0±0.5℃through changing hot-water bottle and regulating theheight of filament lamp.In group S, rat was received the same surgical procedures except that thebilateral vertebral arteries were not electrocauterized and the bilateral commoncarotid arteries were not occluded. In group C, group H, group I and group HI,global cerebral ischemia-reperfusion was produced to establish global cerebralischemia-reperfusion injury model. In group H and group HI, a cotton ball anda suction device was placed in the pharynx to avoid aspiration. A temperatureprobe was placed into the right hippocampal CA1region to monitorhippocampal temperature. Two20G silicone tubes were inserted into bothnasal cavities to apply nasopharyngeal cooling and cold physiological saline(5℃) was infused at a rate of100mL·min-1·kg-1until the hippocampaltemperature was reduced to (33.0±0.5)℃. Then bilateral common carotidarteries were clamped for15min. Hypothermia was maintained for1hfollowed by rewarming spontaneously. In group I and HI, rats were givenIGF-1(5μg) via tail vein at1hour after reperfusion.After rats were observed for8h in group S and reperfused for8h in othergroups, six rats were selected randomly to anesthetize again in each group.Rats were firstly perfused by physiological saline via left ventricle, 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 ofpAkt, pFoxO3a, Bcl-2and Bax protein, and used in HE staining.The other six rats were anesthetized in each group, and then brain tissueswere obtained quickly. Samples were frozen in liquid nitrogen, and werestored at-70℃after1h. These samples were used to observe the expression ofpAkt protein and Akt protein by Western blotting.Results:1The results of light microscopeGroup S: The morphology and structure of neurons were normal in the hippocampal CA1region. The cells were neatly arranged, evenly distributed,with abundant cytoplasm, large nuclei (round or oval) and prominent nucleoli.Group C: 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 chromatin condensation. Swelling cells andthe cell debris could be seen. Group H: Compared with group C,histopathological changes in the hippocampal CA1region were minor ingroup H. The living cells in group H were significantly greater than those ingroup C. Group I: The morphology and structure of neurons were slightlydamaged. The cells were regularly arranged. Compared with group C,thenumber of surviving cells was increased. Group HI: Compared with groupC,H and group B, the extent of damage in the hippocampal CA1neurons wasminimum. The number of surviving cells was significantly increased.2. The comparison of expressions of pAkt, pFoxO3a, Bcl-2and Bax protein inhippocampus CA1region (IHS)Compared with group S, the expression of pAkt, pFoxO3a, Bcl-2andBax protein were all increased in other groups. Compared with group C, theexpression of Bax protein was reduced and in group H, group I and group HI,as well as the expression of pAkt, pFoxO3a, Bcl-2were significantlyincreased. Compared with group HI, the expression of pAkt, pFoxO3a, Bcl-2protein were significantly lower in group C, group H and group I. Thedifferences were statistically significant (P<0.05).There was no statisticallysignificant difference of expression of all proteins between group H and group(P>0.05).3The comparison of expression of pAkt protein and Akt protein inhippocampus CA1region (Western blot)Compared with group S, the expressions of pAkt protein was higher ingroup C, group H, group I and group HI. Compared with group C, theexpression of pAkt protein was higher in group H, group Iand group HICompared with group HI.The expression of pAkt protein was significantly highest in group HI. The differences were statistically significant (P<0.05).There was no statistically significant differences in the comparison ofexpression of Akt protein in hippocampus CA1region (P>0.05).Conclusions：1Mild head hypothermia, IGF-1treatment and their combination can alleviateglobal ischemia-reperfusion injury and have neuroprotective effects in rats.2Combination of IGF-1and mild head hypothermia has more enhancedneuroprotective effect than either used alone.3Mechanisms of the effects of mild head hypothermia or IGF-1are likely toprotect brain from ischemia-reperfusion by inhibiting neuronal apoptosis viaincreasing the phosphorylation of Akt and then pFoxO3a in the downstream. |
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