丰富环境对脑缺血再灌注损伤大鼠神经功能和缺血半暗带区葡萄糖代谢的影响

Effects of Enriched Environment on Neurological Function and Glucose Metabolism in Ischemic Penumbra in Cerebral Ischemia-reperfusion Injury Rats

目的探讨丰富环境对脑缺血再灌注损伤大鼠神经功能及缺血半暗带区葡萄糖代谢的影响。方法成年雄性Sprague-Dawley大鼠72只随机分为假手术组(n=24)、模型组(n=24)和丰富环境组(n=24)。后两组采用改良Longa法制备大鼠脑缺血60 min再灌注模型。丰富环境组造模后立即饲养在丰富环境中。分别于造模前及造模后1 d、7 d、14 d、21 d、28 d采用改良神经损害严重程度评分(mNSS)进行评估。术后1 d和28 d,神经功能评估后各组取12只,HE染色观察脑组织病理结构,逆转录实时定量聚合酶链反应(RT-qPCR)和Western blotting检测缺血半暗带缺氧诱导因子-1α(HIF-1α)、葡萄糖转运蛋白1(GLUT1)、6-磷酸果糖-2-激酶/果糖-2,6-双磷酸酶3(PFKFB3)mRNA和蛋白表达;高效液相色谱法测定缺血半暗带ATP、ADP和AMP变化,计算能量负荷(EC)。结果术后14 d后,丰富环境组mNSS评分持续低于模型组(P<0.05)。术后1 d,与假手术组相比,模型组和丰富环境组缺血半暗带细胞出现水肿、核固缩边缘化、成空泡状排列等病理改变,ATP含量及EC下降(P<0.05),HIF-1α、GLUT1和PFKFB3 mRNA和蛋白表达增高(P<0.05)。术后28 d,与模型组相比,丰富环境组病理改善,缺血半暗带区ATP含量及EC增高(P<0.05),HIF-1α、GLUT1和PFKFB3 mRNA和蛋白表达增高(P<0.05)。结论丰富环境可促进脑缺血再灌注损伤大鼠神经功能恢复,可能与上调HIF-1α及其下游基因GLUT1和PFKFB3表达,改善葡萄糖代谢有关。

Objective To explore the effects of enriched environment on neurological function in cerebral ischemia-reperfusion injury rats and the glucose metabolism in ischemic penumbra.Methods A total of 72 adult male Sprague-Dawley rats were randomly divided into sham group(n=24),model group(n=24)and enriched environment group(n=24).The latter two groups suffered cerebral ischemia 60 minutes and reperfused with modified Longa's method.The enriched environment group was fed in enriched environment after operation.All the rats were assessed with modified Neurological Severity Score(mNSS)before,and one,seven,14,21 and 28 days after operation.One and 28 days after operation,twelve rats from each groups were sacrificed after mNSS assessment,respectively.The histopathology was observed with HE staining.The expressions of hypoxia-inducible factor-1α(HIF-1α),glucose transporter 1(GLUT1)and 6-phosphofructo-2-kinase/fructose-2-bisphosphatase 3(PFKFB3)in ischemic penumbra were determined with reverse transcription realtime quantitative polymerase chain reaction(RT-qPCR)and Western blotting.The levels of ATP,ADP and AMP in ischemic penumbra were measured with high performance liquid chromatography(HPLC)and the energy charge(EC)was calculated.Results Compared with the model group,the scores of mNSS decreased in the enriched environment group since 14 days after operation(P<0.05).The cells in the penumbra presented edema,nuclear pyknosis marginalization,vacuolar arrangement and other pathological changes in the model group and the riched environment group one day after operation;while compared with the model group,the levels of ATP and EC decreased and the mRNAs and protein expression of HIF-1α,GLUT1 and PFKFB3 increased in the enriched environment group(P<0.05).The pathology improved in the riched environment group compared with that in the model group 28 days after operation;while the mRNAs and protein expression of HIF-1α,GLUT1 and PFKFB3 increased,as well as the levels of ATP and EC(P<0.05).Conclusion Enriched environment can promote the recovery of neurological function in rats after cerebral ischemia-reperfusion,which may associate with promoting expression of HIF-1αand downstream GLUT1 and PFKFB3,and improving glucose metabolism.