胶质细胞在清醒自由移动小鼠缺血损伤后的动态变化

Dynamic changes of glial cells in conscious freely moving mice after photothrombosis-induced ischemia

目的：探讨星形胶质细胞、小胶质细胞在清醒自由移动小鼠脑缺血损伤后的动态变化、神经发生和运动功能受损情况。方法：采用光化学法建立局灶性光栓缺血模型,用Nissl染色、Brd U标记和免疫荧光检测缺血损伤后不同时间点的缺血损伤面积、细胞增殖数量、胶质细胞数量改变及神经发生情况,并通过旷场实验检测缺血损伤后的行为学变化。结果：Nissl染色显示细胞缺损面积在2 d内达到最高峰,其后逐渐减小,在30 d后基本恢复。损伤后3～4 d Brd U标记的新生细胞数目达到最大,这与缺血区GFAP阳性的星形胶质细胞和Iba-1阳性的小胶质细胞的数量增加相一致。在缺血损伤后第6 d纹状体内出现微管相关蛋白（doublecortin,DCX）阳性细胞,暗示存在短暂的神经发生事件。缺血损伤导致的纹状体功能障碍最严重时间在缺血后的第2 d,之后减弱但持续存在。结论：光栓缺血损伤后第2～4 d是细胞增殖、行为功能障碍、胶质细胞聚集的最显著期,之后有短暂的神经发生。损伤后胶质细胞与神经发生的时间顺序可能为利用胶质细胞转分化为神经元治疗缺血性脑损伤提供线索。

Objective： Exploring dynamic changes in astrocytes,microglia,neurogenesis and behavioral deficit in awake and free moving mice with local ischemic injury. Methods： Photochemical method was used to establish focal ischemia after photothrombosis. Nissl staining,Brd U labeling combined with immunofluorescence was used to detect ischemic area,observe proliferation and distribution of glia cells,immunostaining of doublecortin（ DCX） was used to show local neurogenesis,and open field test was used to detect locomotion deficit post-ischemic injury. Results： Nissl staining showed that ischemic damage area reached the peak in two days,then gradually decreased and appeared to be recovered30 d post ischemia. The maximal number of proliferating cells labeled with Brd U in ischemic area was observed 3 ~ 4 d after injury,and GFAP-positive astrocytes and Iba-1-positive microglia displayed similar dynamic changes in terms of cell number. An obvious appearance of DCX-positive neuroblasts was observed 6 d post ischemia suggesting a transient neurogenesis in ischemic area. The most serve striatal dysfunction caused by ischemic damage was observed in the first 2 d after ischemia,and the behavioral deficit was reduced but persistent until 30 d post ischemia. Conclusion： The first 2 ~ 4 d post ischemia is a key period during which a drastic cellular proliferation,increase of glia cells and behavioral deficits arepresent,while obvious neurogenesis in the ischemic striatum is observed a week after ischemia. This time window in the proliferation of glia cells and presence of neuroblasts may be used to repair brain damage by using the transformation of glia cells into neurons with the help of genetic manipulation and / or approaches.