大鼠脑梗死后缺氧组织显像的动态变化

【目的】 研究大鼠脑梗死后缺氧组织是否能长期存在及其动态变化。 【方法】 利用大鼠大脑中动脉闭塞(MCAO)模型,分别采用99Tcm-HL91放射自显影和EF5免疫荧光的方法进行缺氧显像。分持续缺血组(PI)、1.5 h缺血再灌注组(1.5 h IR)、2 h缺血再灌注组和3 h缺血再灌注组4组,放射自显影的观察时间点选取手术后1 d和14 d,免疫荧光则选取术后1、3、7、14、21 d,每个时间点各3只大鼠。【结果】 放射自显影显示1.5 h IR组手术后1 d和14 d均为缺氧阳性,其余各组均阴性;免疫荧光示1.5 h IR组术后1、3、7和14 d均有缺氧组织,面积(um2)有缩小倾向(分别为21 478 ± 8 254、 4 405 ± 1 490、2 647 ± 463和1 612 ± 239),但荧光强度没有明显减弱(分别为1.66 ± 0.07、1.75 ± 0.02、1.68 ± 0.08和1.64 ± 0.01);而其余各组在术后7 d均已无缺氧组织。【结论】 与人类脑梗死相似,大鼠脑梗死后缺氧组织可存在较长时间,且随着时间的延长而逐渐减少;1.5 h IR的大鼠MCAO模型是研究脑梗死后缺氧组织长时间存在的合适模型。     

大鼠脑梗死后乏氧组织长时间存在与星形胶质细胞的关系

目的 探讨大鼠脑梗死后乏氧组织的长时间存在与星形胶质细胞的关系.方法 利用大鼠脑缺血1.5 h再灌注(1.5 h IR组)和持续缺血(PI组)的大脑中动脉闭塞模型,采用EF5和胶质纤维酸性蛋白(GFAP)免疫荧光双标方法观察乏氧组织和星形胶质细胞增殖活化情况,比较两组术后1、3、7、14d缺血侧大脑半球皮层的星形胶质细胞GFAP荧光强度以及与乏氧组织的关系.结果 1.5 h IR组术后1、3、7、14 d均见乏氧组织存在,PI组乏氧组织仅存在3 d.各观察时间点乏氧组织内的GFAP荧光强度均较周围区域高,差异有统计学意义(P<0.05).随着时间的延长,两组的GFAP荧光强度均不断增强,在术后7 d达到高峰,14 d时下降,差异有统计学意义(P<0.05);且各时间点的1.5 h IR组的GFAP荧光强度均高于PI组,差异有统计学意义(P<0.05).结论 脑梗死后星形胶质细胞在乏氧组织内增殖活化尤为明显,乏氧组织的长时间存在与星形胶质细胞密切相关.     

氧葡萄糖剥夺-再恢复小鼠小胶质细胞的激活及Toll样受体9表达的变化

目的观察氧葡萄糖剥夺-再恢复(OGDR)对小鼠BV-2小胶质细胞的激活以及Toll样受体9(TLR9)表达的影响。方法采用OGDR法建立BV-2细胞缺氧、缺糖模型,以常氧培养的BV-2细胞作为对照。使用倒置相差显微镜观察BV-2细胞在OGDR后0、6、12、24、48、72 h形态的变化,CCK8法检测OGDR后不同时间点细胞存活率的变化,反转录PCR和Western Blot检测细胞内TLR9 mRNA和蛋白的表达。结果①OGDR后BV-2细胞由原来静止的分枝状变成激活的阿米巴状。②OGDR后,细胞存活率明显下降,0 h为对照组的(65.7±9.2)%;12 h下降至最低,为对照组的(44.8±2.3)%,后逐渐升高,48、72 h分别为对照组的(60.8±10.2)%、(72.3±10.0)%。③OGDR后,随着时间的延长,TLR9 mRNA表达逐渐升高,24 h达高峰,后逐渐降低,但72 h仍高于0 h时间点的表达。TLR9蛋白表达亦呈升高趋势,在72 h达高峰。对照组各时间点TLR9 mRNA、TLR9蛋白表达差异均无统计学意义。④相同时间点的比较,OGDR组OGDR后6～72 h,TLR9 mRNA表达均明显高于对照组(P<0.05,或P<0.01,);12～72 h,TLR9蛋白表达显著高于对照组(P<0.05,或P<0.01)。结论 OGDR后BV-2细胞被激活,其细胞内TLR9表达随着时间的延长逐渐增高,可能在脑缺血-再灌注后的炎性反应中,发挥重要作用。     

功能磁共振成像对急性脑梗死患者预后判断的价值

目的探讨功能磁共振成像(functional magnetic resonance imaging,fMRI)检测脑感觉运动皮质(sensorimotor cortex,SMC)区激活对于急性脑梗死患者预后的评估价值。方法对44例脑梗死患者在起病6～72 h内和14 d进行手被动运动模式的fMRI成像以及美国国立卫生院卒中量表(NIHSS)评分,在起病30 d和90 d时进行修正Barthel指数(modified Barthel index,MBI)评分和修正Rankin量表(modified Rankin scale,mRS)评分。结果 SMC激活组患者的NIHSS评分低于无激活组(6～72 h:9.5±2.1 vs 11.9±4.2,P=0.023;14 d:中位数:5 vs 10,P=0.01),脑梗死体积小于无激活组(P<0.05);14 d SMC激活组的30 d和90 d MBI、30 d mRS评分均优于无激活组(P<0.05),14 d SMC激活体积与14 d NIHSS、30 d和90 d的MBI、mRS评分值也有相关关系(P<0.05)。结论手被动运动激活SMC区的fMRI成像对急性脑梗死患者有预后评估价值,尤其起病14 d时SMC区的激活情况与患者预后密切相关。     

脑缺血再灌注大鼠模型中乏氧脑组织的长时程存在

BACKGROUND： Hypoxic tissue surrounding the ischemic core may represent the ischemic penumbra following cerebral infarction. However, some studies have shown that the duration of ischemic tissue is longer than previously believed. OBJECTIVE： To clarify whether cerebral hypoxic tissue could survive long-term and whether it is altered in rats following cerebral infarction; to establish an ischemiaJreperfusion model in which hypoxic tissue exists for extended periods of time. DESIGN, TIME AND SE＇I-I＇ING： A completely randomized grouping and controlled experiment was performed at the Experimental Animal Center of Sun Yat-sen University and Medical Research Center, the Second Affiliated Hospital of Sun Yat-sen University between June and December 2008. MATERIALS： 4,9-diaza-3,3,10,10-tetramethyldodecan-2, 11 -dione dioxime （BnAO） （HL91）, used as the hypoxic marker for autoradiography, was supplied by the Beijing Syncor Star Medicinal, China, and the flesh eluent Na99TcmO4 to mark HL91 was supplied by Guangzhou Medical Isotope Center of the China Institute of Atomic Energy. 2-（2-nitro-1H-imidazole-l-yl）-N-（2,2,3,3,3-pentafluoropropyl） acetamide （EF5） and its antibody ELK3-51, used as a hypoxic marker for immunofluorescence, were supplied by the University of Pennsylvania, USA. METHODS： Male Sprague Dawley rats were randomly divided into four groups： 1.5-hour ischemiaJreperfusion group （1.5 h IR）, 2-hour ischemiaJreperfusion group （2 h IR）, 3-hour ischemiaJreperfusion group （3 h IR）, and permanent ischemia （PI） group, with 21 rats in each group. The middle cerebral artery occlusion model was established using the intraluminal suture method, while reperfusion was performed by removing the suture at each observation time point. However, in the PI group, the suture was left in the artery. MAIN OUTCOME MEASURES： Area and average absorbance of fluorescence, representing hypoxic tissue, were measured by image-analysis. RESULTS： Autoradiography revealed positive hypoxia at days 1 and 14 postoperatively in the 1.5 h IR group. Immunohistochemistry results demonstrated that hypoxic tissue existed in the 1.5 h IR group on days 1, 3, 7, and 14, with decay of the area, but no significant weakening of fluorescent intensity. Hypoxic tissues were not observed at day 7 postoperatively in the 2 h and 3 h IR groups, as well as PI group. CONCLUSION： Similar to human cerebral infarction, hypoxic tissues in rats exist for an extended period of time following cerebral infarction, and diminish over even longer periods. Moreover, the 1.5 h IR rat model was the suitable model for studying long-term hypoxic tissue after cerebral infarction.