慢性脑缺血后脑组织AQP4的表达变化

Change in the expression of aquaporin-4 in brain tissue after chronic cerebral ischemia

目的:探讨水通道蛋白-4(aquaporin-4,AQP4)在慢性脑缺血后的表达变化及其可能作用。方法:将40只SD大鼠随机分为对照组(N组,10只)和实验组(30只);实验组根据脑缺血时长,分为慢性脑缺血2周组(2W组)、1月龄组(1M组)和2月龄组(2M组),每组各10只。采用双侧颈总动脉永久结扎法(2-VO),构建大鼠慢性脑缺血模型。干湿重法检测大鼠脑含水量,苏木精-伊红染色法观察大鼠脑组织形态学的变化,尼氏染色法观察细胞凋亡情况。免疫荧光法检测AQP4的表达水平和分布情况,免疫印迹法测定AQP4蛋白的相对表达量。免疫荧光检测AQP4与小胶质细胞/巨噬细胞特异性蛋白抗体(ionized calcium binding adapter molecule 1,IBA1)(小胶质细胞的标志物)表达情况。结果:大鼠慢性脑缺血组脑含水量(77.778±0.042,77.813±0.142,77.805±0.027)与对照组(77.786±0.029)相比,无统计学差异(F=0.136,P=0.936),且慢性脑缺血各组之间亦无统计学差异。苏木精-伊红染色和尼氏染色结果显示:与对照组相比,慢性脑缺血组脑组织细胞排列紊乱,部分细胞核固缩、消失,尼氏体数量减少。免疫荧光结果显示,慢性脑缺血组大鼠脑组织较对照组AQP4表达增强(前额叶:F=1 089.311,P=0.000,N组:90.000±1.000,2W组:166.722±3.660,1M组:200.347±0.284,2M组:229.333±5.033;顶叶:F=784.332,P=0.000,N组:171.512±1.340,2W组:215.107±1.817,1M组:223.014±2.080,2M组:232.654±1.319),小胶质细胞活化增生,且部分AQP4与IBA1共标(前额叶:F=182.218,P=0.000,N组:0.718±0.012,2W组:0.822±0.000,1M组:0.907±0.016,2M组:0.970±0.020;顶叶:F=785.416,P=0.000,N组:0.861±0.009,2W组:0.912±0.005,1M组:0.966±0.003,2M组:0.751±0.003);免疫印迹法结果显示,慢性脑缺血组大鼠脑组织AQP4的表达水平上调,并且随缺血时间延长,其表达进一步增强(前额叶:F=587.102,P=0.000,N组:0.589±0.026,2W组:0.938±0.028,1M组:1.185±0.011,2M组:1.515±0.060;顶叶:F=86.881,P=0.000,N组:0.663±0.073,2W组:0.865±0.044,1M组:1.228±0.082,2M组:1.282±0.047)。结论:慢性脑缺血后,由于缺血缺氧导致神经元凋亡、小胶质细胞活化增生和AQP4表达上调,上调的AQP4可能参与了慢性脑缺血后的相关炎症过程。

Objective:To investigate the change in the expression of aquaporin-4(AQP4)after chronic cerebral ischemia and the possible role of AQP4. Methods:A total of 40 Sprague-Dawley rats were randomly divided into control group(N group with 10 rats)and experimental group with 30 rats;according to the duration of cerebral ischemia,the experiment group was further divided into 2-week chronic cerebral ischemia group(2W group with 10 rats),1-month-old group(1M group with 10 rats),and 2-month-old group(2M group with 10 rats). Ligation of both common carotid arteries was performed to establish a rat model of chronic cerebral ischemia. The wet-dry weight method was used to measure brain water content,hematoxylin and eosin staining was used to observe the histological changes of brain tissue,and Nissl staining was used to observe cell apoptosis. Immunofluorescence assay was used to measure the expression and distribution of AQP4,and Western blot was used to measure the relative expression level of AQP4 protein. Immunofluorescence assay was used to measure the expression of AQP4 and IBA1,a marker for microglial cells. Results:There was no significant difference in brain water content between the 2W/1M/2M groups and the N(77. 778 ± 0. 042/77. 813 ±0. 142/77.805±0.027 vs. 77.786 ±0.029,F=0.136,P=0.936),and there was no significant difference between the 2W,1M,and 2M groups. Hematoxylin and eosin staining and Nissl staining showed that compared with the N group,the experimental group had disordered arrangement of brain cells,karyopyknosis or disappearance of nuclei in some cells,and a reduction in Nisslbodies. Immunofluorescence assay showed that compared with the N group,the 2W,1M,and 2M groups had significantly higher expression of AQP4 in the prefrontal lobe(166.722±3.660/200.347 ±0.284/229.333 ±5.033 vs. 90.000 ±1.000,F =1 089.311,P=0.000)and the parietal lobe(215.107±1.817/223.014±2.080/232.654±1.319 vs. 171.512±1.340,F=784.332,P=0.000),as well as activation and proliferation of microglial cells,and AQP4 was partially co-labeled with IBA1(prefrontal lobe:0.822 ±0.000/0.907±0.016/0. 970 ± 0. 020 vs. 0.718±0.012,F=182.218,P=0.000;parietal lobe:0.912±0.005/0.966±0.003/0.751±0.003 vs. 0.861±0.009,F=785.416,P=0.000). Western blotting showed that compared with the N group,the 2W,1M,and 2M groups had a significant increase in the expression of AQP4 in brain tissue over the time of ischemia(prefrontal lobe:0.938±0.028/1.185±0.011/1.515±0.060 vs. 0.589 ±0.026,F=587.102,P=0.000;parietal lobe:0.865±0.044/1.228±0.082/1.282±0.047 vs. 0.663±0.073,F=86.881,P=0.000). Conclusion:After chronic cerebral ischemia,hypoxia and ischemia may cause neuronal apoptosis,activation and proliferation of microglial cells,and upregulation of AQP4 expression,suggesting that AQP4 may be involved in the inflammatory process after chronic cerebral ischemia.