大鼠脑缺血与缺血再灌注介导的神经元损伤
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摘要
目的通过分别建立SD大鼠完全脑缺血和脑缺血再灌注模型,比较研究完全脑缺血和脑缺血再灌注介导的神经元损伤,以及相关炎症因子的表达。方法将成年雄性SD大鼠(42只,体重250～350 g)随机分为7组,构建脑缺血24、48、72 h模型及脑缺血2 h再灌注24、48、72 h模型。以Longa评分标准对大鼠进行神经功能评分;2,3,5-氯化三苯基四氮唑(2,3,5-triphenyltetrazolium chloride,TTC)染色测定脑梗死体积;苏木精-伊红(hematoxylin-eosin,HE)染色检测大脑皮层、海马CA3区神经元损伤程度;免疫荧光法检测NOD样受体蛋白3(NOD-like receptor protein 3,NLRP3,又称NALP3)在脑组织中的表达及定位。结果与正常组比较,完全脑缺血和脑缺血再灌注模型大鼠的Longa评分更高,且脑组织梗死体积显著增大。完全脑缺血和脑缺血再灌注模型大鼠大脑皮层和海马CA3区的神经元损伤明显;随着再灌注时间的延长,再灌注48 h大鼠的脑皮层和海马CA3区神经元损伤明显减轻,但在再灌注72 h时再度加重。与正常组比较,完全缺血24 h和缺血2 h再灌注24 h后,NALP3-Cy3荧光表达明显增强。结论完全脑缺血和脑缺血再灌注均可造成大脑皮层和海马CA3区的神经元损伤,仅在缺血2 h再灌注48 h时大脑损伤可部分恢复;在上述两模型中炎症因子NALP3的表达均显著升高。
Objective Models of complete cerebral ischemia and cerebral ischemia reperfusion in SD rats were established to compare and study the neuronal cell damage and the expression of related inflammatory factors.Methods Adult male SD rats(80 rats,weighing 250-350 g)were randomly divided into seven groups.Longa scores were used to assess neurological function in rats.TTC staining was used to measure the volume of cerebral infarction.HE staining was used to detect the degree of neuronal damage in cerebral cortex and hippocampal CA3 region.Immunofluorescence was used to detect the expression level and expression site of NALP3 protein.Results The Longa score was higher in the models of complete cerebral ischemia and cerebral ischemia reperfusion than in the normal group.Meanwhile,the infarct size of the brain tissue was significantly increased.The results of HE staining showed that rats with complete cerebral ischemia and rats with cerebral ischemia-reperfusion had significant neuronal damage in the cerebral cortex and hippocampal CA3 region.Moreover,with the extension of reperfusion time,neuronal damage in the cortex and hippocampal CA3 area of rats was significantly reduced 48 h after reperfusion,but re-emerged 72 h after reperfusion.Immunofluorescence analysis indicated that the expression of NALP3-Cy3 was significantly increased in the 24 h complete ischemia group and 2 h ischemia/24 h reperfusion group,compared with the normal group.Conclusion Complete cerebral ischemia and cerebral ischemia reperfusion can mediate neuronal damage in the cerebral cortex and hippocampal CA3 region,and brain damage can be partially restored only in 2 h ischemia/48 h reperfusion group.The expression of the inflammatory factor NALP3 is significantly increased in both constructed models.
Objective Models of complete cerebral ischemia and cerebral ischemia reperfusion in SD rats were established to compare and study the neuronal cell damage and the expression of related inflammatory factors.Methods Adult male SD rats(80 rats,weighing 250-350 g)were randomly divided into seven groups.Longa scores were used to assess neurological function in rats.TTC staining was used to measure the volume of cerebral infarction.HE staining was used to detect the degree of neuronal damage in cerebral cortex and hippocampal CA3 region.Immunofluorescence was used to detect the expression level and expression site of NALP3 protein.Results The Longa score was higher in the models of complete cerebral ischemia and cerebral ischemia reperfusion than in the normal group.Meanwhile,the infarct size of the brain tissue was significantly increased.The results of HE staining showed that rats with complete cerebral ischemia and rats with cerebral ischemia-reperfusion had significant neuronal damage in the cerebral cortex and hippocampal CA3 region.Moreover,with the extension of reperfusion time,neuronal damage in the cortex and hippocampal CA3 area of rats was significantly reduced 48 h after reperfusion,but re-emerged 72 h after reperfusion.Immunofluorescence analysis indicated that the expression of NALP3-Cy3 was significantly increased in the 24 h complete ischemia group and 2 h ischemia/24 h reperfusion group,compared with the normal group.Conclusion Complete cerebral ischemia and cerebral ischemia reperfusion can mediate neuronal damage in the cerebral cortex and hippocampal CA3 region,and brain damage can be partially restored only in 2 h ischemia/48 h reperfusion group.The expression of the inflammatory factor NALP3 is significantly increased in both constructed models.
引文
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[24] 赵新玲,陶然,杨园,等.血小板反应蛋白1(THBS-1)与急性缺血性脑卒中诊断和预后的相关性研究[J].华中科技大学学报:医学版,2018,47(5):594-598.
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[2] Pantoni L,Sarti C,Inzitari D.Cytokines and cell adhesion molecules in cerebral ischemia experimental bases and therapeutic perspectives[J].Arterioscler Thromb Vasc Biol,1998,18(4):503-513.
[3] 张增利.雌激素新型受体GPR30调控TLR4抑制小胶质细胞炎症反应在脑缺血损伤中的保护作用[D].西安:第四军医大学,2017:3-10.
[4] Lively S,Schlichter L C.Age-related comparisons of evolution of the inflammatory response after intracerebral hemorrhage in rats[J].Transl Stroke Res,2012,3(Suppl 1):132-146.
[5] 陆士奇,冀勇,刘春风.NF-κB与脑缺血脑缺氧再灌注损伤[J].中国急救医学,2005,25(4):279-281.
[6] Zhang C,Boini K M,Xia M,et al.Activation of nod-like receptor protein 3 inflammasomes turns on podocyte injury and glomerular sclerosis in hyperhomocysteinemia[J].Hypertension,2012,60(1):154.
[7] 郝洁芦.NALP3在大鼠肾缺血再灌注损伤中的表达及作用研究[D].太原:山西医科大学,2012:36-39.
[8] 王娜梅,赵玉红,解旭东.缺血性脑血管病的治疗现状及进展研究[J].医学综述,2017,23(19):3822-3826.
[9] 熊维,常飞.脑心通胶囊联合丹红注射液治疗急性脑梗死的临床疗效及其机制探讨[J].华中科技大学学报:医学版,2016,45(1):74-77.
[10] Johann S,Beyer C.Neuroprotection by gonadal steroid hormones in acute brain damage requires cooperation with astroglia and microglia.[J].J Steroid Biochem Mol Biol,2013,137(18):71-81.
[11] Muren M A,Hütler M,Hooper J.Functional capacity and health-related quality of life in individuals post stroke[J].Top Stroke Rehabil,2008,15(1):51-58.
[12] 马夏珍.基于核磁共振技术研究脑缺血再灌注大鼠脑皮质代谢物组水平变化的分子机制[J].第二军医大学学报,2016,37(11):1338-1347.
[13] Steiner E,Enzmann G U,Lin S,et al.Loss of astrocyte polarization upon transient focal brain ischemia as a possible mechanism to counteract early edema formation[J].Glia,2012,60(11):1646-1659.
[14] 王晓平,倪京满.脑缺血再灌注损伤的研究及药物治疗进展[J].中国新药杂志,2016,25(6):659-663.
[15] Sakatani K,Iizuka H,Young W.Somatosensory evoked potentials in rat cerebral cortex before and after middle cerebral artery occlusion[J].Stroke,1990,21(1):124.
[16] Di Lazzaro V,Pellegrino G,Di Pino G,et al.Val66 Met BDNF gene polymorphism influences human motor cortex plasticity in acute stroke[J].Brain Stimul,2015,8(1):92-96.
[17] Zhang H Q,Li P F,Li S Y,et al.Influence of lactuside B on the expression of AQP4 and TRPM7 mRNAs in the cerebral cortex after cerebral ischemia injury[J].Eur Rev Med Pharmacol Sci,2014,18(8):1151-1157.
[18] 李占标,刘方铭,刘维菊.针刺疗法对局灶性脑缺血大鼠神经功能、大脑皮层蛋白激酶A表达及细胞凋亡率的影响[J].针刺研究,2013,38(2):106-111.
[19] Zheng W X,Cao X L,Wang F,et al.Baicalin inhibiting cerebral ischemia /hypoxia-induced neuronal apoptosis via MRTF-A-mediated transactivity[J].Eur J Pharmacol,2015,767:201-210.
[20] 张天舒,阮志,刘霞,等.MCAO大鼠脑缺血再灌注损伤机制的核磁共振代谢组学研究[J].中国药科大学学报,2016,47(2):188-198.
[21] Broderick J P,Palesch Y Y,Demchuk A M,et al.Endovascular therapy after intravenous t-PA versus t-PA alone for stroke[J].N Eng J Med,2013,368(10):893-903.
[22] Wang Y,Wang Y,Li M,et al.1H NMR-based metabolomics exploring biomarkers in rat cerebrospinal fluid after cerebral ischemia/reperfusion[J].Mol BioSys,2013,9(3):431-439.
[23] 万东.梓醇上调局灶脑缺血大鼠缺血灶周围大脑皮质BDNF和TrkB蛋白表达[J].中国药理学通报,2013,29(6):787-792.
[24] 赵新玲,陶然,杨园,等.血小板反应蛋白1(THBS-1)与急性缺血性脑卒中诊断和预后的相关性研究[J].华中科技大学学报:医学版,2018,47(5):594-598.
[25] Zhang N,Zhang X,Liu X,et al.Chrysophanol inhibits NALP3 inflammasome activation and ameliorates cerebral ischemia/reperfusion in mice[J].Mediators Inflamm,2014,2014:370530.
[26] Zhu P,Duan L,Chen J,et al.Gene silencing of NALP3 protects against liver ischemia-reperfusion injury in mice[J].Human Gene Therapy,2011,22(7):853-864.
[27] 张楠.NALP3炎症复合体在脑缺血再灌小鼠脑组织中的动态表达及大黄酚的神经保护作用[D].石家庄:河北医科大学,2014:3-6.
[28] Mariathasan S,Weiss D S,Newton K,et al.Cryopyrin activates the inflammasome in response to toxins and ATP[J].Nature,2006,440(7081):228-232.
[29] Willingham S B,Bergstralh D T,O′Connor W,et al.Microbial pathogen-induced necrotic cell death mediated by the inflammasome components CIAS1/Cryopyrin/NLRP3 and ASC[J].Cell Host Microbe,2007,2(3):147-159.