不同疗程高压氧对缺氧缺血性脑损伤新生大鼠神经干细胞增殖和分化的影响

Effect of Different Courses Hyperbaric Oxygen Treatment on Proliferation and Differentiation of Neural Stem Cells in Hypoxic-Ischemic Brain Damage Rats

目的探讨不同疗程高压氧(HBO)对缺氧缺血脑损伤(HIBD)新生大鼠神经干细胞(NSCs)增殖和分化的影响。方法7日龄SD大鼠随机分为正常对照组(CON组)、HIBD组、HBO组,其中HBO组又分为HBO1疗程组[HBO-1,在HIBD2 h开始HBO治疗,HBO压力为0.1 MPa(2 ATA),稳压60 min,1次/d,7次为1疗程],2疗程组(HBO-2,予2个疗程HBO)和3疗程组(HBO-3,予3个疗程HBO)。疗程结束后(鼠龄35 d)取脑组织标本,采用5-溴-2-脱氧嘧啶核苷(BrdU)/神经巢蛋白(Nestin)免疫荧光双标法检测缺血侧侧脑室室管膜下区(SVZ区)和海马齿状回区(DG区)的内源性NSCs的变化。Western blot法检测缺血侧大脑半球Nes-tin蛋白变化。BrdU/神经元特异性烯醇化酶(NSE)和BrdU/胶质纤维酸性蛋白(GFAP)免疫荧光双标法检测大脑皮层神经元和星形胶质细胞数量;TUNEL法检测皮层神经细胞凋亡;并进行T迷宫试验、放射臂迷宫测试、足错误等行为学测试。结果1.HBO治疗后SVZ区BrdU+/Nestin+细胞随疗程增加而逐渐增加,以HBO-3组的阳性细胞数最多;2.各组大鼠的海马DG区均仅有极少量的BrdU+/Nestin+细胞表达,且随着HBO疗程的增加,BrdU+/Nestin+细胞表达无增加;3.大脑皮层BrdU+/NSE+细胞表达随HBO疗程增加逐渐增多,其中以HBO-3组的阳性细胞数最多;4.HIBD组可见较多GFAP+细胞,高于CON组和HBO组;且各组均仅有个别BrdU+/GFAP+细胞表达;5.Western blot显示HBO-3组Nestin蛋白表达高于其余各组;6.T迷宫、放射臂迷宫和足错误测试结果均表明随着HBO疗程的增加,疗效愈加显著。结论增加HBO疗程可促进HIBD新生大鼠SVZ区内源性NSCs皮层神经元细胞的增殖和分化。

Objective To explore the effect of different courses hyperbaric oxygen （HBO） promote proliferation and differentiation of neural stem cells（NSCs） in hypoxic - ischemic brain damage（HIBD） newborn rats. Methods Seven - day - bid SD rats were subjected to unilateral carotid ligation followed by 2 hours of hypoxia （80 mL/L 02 ）. Difference Courses of HBO were administered after 2 hours later （2 ATA, once daily for 7 days a course ）. At the rats＇ age of 35 days, BrdU/Nestin immunofluorescence in the injury subventricular zone （SVZ） and dentate gyrus （DG）, BrdU/NSE, BrdU/GFAP immunofluorescence in the injury brain cortex and Nestin protein were detected through Western blot analysis. Meanwhile the other pups were tested with T - maze forced alternation, the radial arm maze and foot - fault. Results 1. The number of BrdU^＋/Nestin^＋ cells in SVZ of the HBO treatment groups increased along with the course of HBO, especially in group HBO -3 ;2. There was no obvious increase of BrdU^＋/Nestin^＋ cells in DG of HBO treatment groups ;3. The number of BrdU^＋/NSE^＋ cells in cortex increased along with the courses of HBO;4. The number of GFAP^＋ cells in HIBD was higher than that in other groups,but there were few BrdU^＋/GFAP^＋ cells in all groups ;5. Western blot showed the expression of Nestin protein in group HBO -3 was higher than that in other groups;6. The HBO treatment groups performed better in the behavioral tests along with the courses. Conclusions Multiple courses of HBO treatment may promote neurogenesis of the endogenous NSCs in SVZ and neurons in cortex of perinatal HIBD rats.