目的 探讨芹菜素对急性局灶性脑缺血再灌注损伤大鼠半暗区内小胶质细胞的影响及意义。方法 采用改良线栓法建立大鼠局灶性脑缺血再灌注模型。动物随机分为假手术组、模型组、芹菜素组。模型组和芹菜素组按再灌注时间不同又分为再灌注6、24、48、72 h及7 d各5个小组,共11组。各组观察至规定的时间进行神经行为学评分,其中随机取6只大鼠,进行异硫氰酸荧光素标记的同工凝集素B4(FITC-ILB4)染色,激光扫描共聚焦显微镜(LSCM)观察浸润的单核细胞及半暗区小胶质细胞形态及数量的变化,余下6只大鼠取脑组织,测定脑含水量。结果 脑缺血再灌注6 h,模型组大鼠脑缺血半暗区周围出现同工凝集素B4阳性(ILB4+)表达细胞即分枝状小胶质细胞(罗丹明6G标记阴性即Rhod 6G?)和浸润的单核细胞(罗丹明6G标记阳性即Rhod 6G+);48 h小胶质细胞明显增多,形态向阿米巴样转变;72 h达到高峰,主要为脑源性小胶质细胞增生,形态以阿米巴样为主;7 d小胶质细胞减少;芹菜素组相应时间点小胶质细胞较模型组明显减少(P<0.05、0.01),形态亦呈现相似改变。脑缺血48、72 h,芹菜素组大鼠脑组织含水量明显低于模型组(P<0.01)。模型组神经行为学评分与半暗区ILB4+细胞数呈负性相关(r=?0.415,P<0.05),且芹菜素能有效降低模型组神经功能缺损程度(P<0.05),明显减轻脑损伤程度。结论 缺血后的大脑脑实质内一部分小胶质细胞来源于循环中的单核细胞;半暗区小胶质细胞形态及数量的变化与脑损伤程度具有相关性;芹菜素对急性局灶性大鼠脑缺血再灌注损伤具有保护作用,且可能与下调小胶质细胞数量,抑制小胶质细胞过度活化密切相关。

Objective To study the effect of apigenin on the expression of microglia in penumbra and cerebral water content after acute transient focal cerebral ischemia-reperfution injury in rats. Methods The acute transient focal cerebral ischemia-reperfusion models in rats were established with the modified suture method. Male Sprague-Dawley rats were randomly divided into Sham group, model group, and apigenin groups (there were 6, 24, 48, 72 h, and 7 d reperfusion treatments in the model and apigenin groups , n =12). All of them are 11 groups. The neurological behavior scores were valued. By FITC labeled isolectin B4 (FITC-ILB4) histochemistry staining, the infiltration of monocytes and the changes of cell morphology and number of brain-derived microglia in penumbra of six rats in every group were observed under laser scanning confocal microscope (LSCM). Water content was measured in isolated brain tissue of other six rats. Results The positive cells of ILB4 (ILB4+) including microglia cells (Rhod 6G?) and infiltration of monocytes (Rhod 6G+) were found in cerebral ischemic area around penumbra of rats after 6 h ischemia-reperfusion in model group; The morphology changed to Amoeba-like; Microglia increased significantly after 48 h and reached to peak in 72 h, which mainly belonged to the proliferation of brain-derived microglia in Amoeba-like morphology. Microglia cell decreased in 7 d, and microglia in apigenin group obviously decreased more than that in model group (P<0.05, 0.01) with the similar morphological change in corresponding time points. In 48 and 72 h of cerebral ischimia, the water content in brain tissue of rats in apigenin group was markedly lower than that in model (P<0.01). There was negative line correlation between the neurological behavior score and the number of ILB4+ cells in penumbra of model group (r＝?0.415, P<0.05). Apigenin could reduce the degree of neurological deficiency in model group and mitigate the brain injury effectively. Conclusion A part of microglia cells inpenumbra are associated with brain injury; Apigenin shows the protection on acute transient focal cerebral ischemia-reperfution injury in rats, which maybe relates with down-regulating the microglia cell number and inhibiting the excessive activation of microglia cell.

温州市科技局国际合作项目(H20070034)