高压氧对局灶性脑缺血大鼠内源性神经干细胞增殖的影响

Effect of hyperbaric oxygen on proliferation of endogenous neural stem cells in rats with focal cerebral ischemia

目的观察不同时期不同疗程高压氧(HBO)对局灶性脑缺血大鼠脑室室管膜下区(SVZ)、海马齿状回(DG)内源性神经干细胞(NSCs)增殖的影响。方法采用线栓法制备大脑中动脉阻塞(MCAO)局灶性脑缺血再灌注模型。将105只大鼠按抽签法随机分为假手术组(SS)、缺血再灌注模型组(IR)、缺血再灌注模型后1 d HBO治疗组(IR1d+HBO)、缺血再灌注模型后7 d HBO治疗组(IR7d+HBO)、缺血再灌注模型后14 d HBO治疗组(IR14d+HBO)和缺血再灌注模型后28 d HBO治疗组(IR28d+HBO)。造模后第2、4、6、8周分别采用免疫荧光BrdU/Nestin双标法检测SVZ、DG区内源性NSCs。结果DG BrdU/Nestin阳性细胞数在造模后2周时,SS组21.20±2.58,IR组56.40±4.51,IR1d+HBO组82.80±7.19,IR7d+HBO组70.00±5.09;4周时SS组21.00±2.92,IR组37.20±3.27,IR1d+HBO组70.60±5.12,IR7d+HBO组57.20±3.56,IR14d+HBO组45.80±4.32;6周时SS组20.20±1.92,IR组26.40±2.74,IR1d+HBO组48.00±3.16,IR7d+HBO组40.60±3.36,IR14d+HBO组31.60±2.41,IR28d+HBO组26.60±2.30;除第8周外,同期各组间DG BrdU/Nestin阳性细胞数差异均有统计学意义(P<0.01)。进一步两两比较显示,与SS组比较,各时间点IR组大鼠DG BrdU/Nestin阳性细胞数均增多(P<0.05);除第8周外,各时间点IR1d+HBO、IR7d+HBO及IR14d+HBO组大鼠DG BrdU/Nestin阳性细胞数均多于IR组(P<0.05);各组均在第2周/最初测量点时DG BrdU/Nestin阳性细胞数达到最多(P<0.05)。SVZ BrdU/Nestin阳性细胞数在造模后2周时,SS组25.20±2.86,IR组66.40±2.96,IR1d+HBO组90.40±6.50,IR7d+HBO组75.00±4.58;4周时SS组24.20±1.48,IR组49.80±4.32,IR1d+HBO组72.40±4.92,IR7d+HBO组57.80±6.46,IR14d+HBO组43.80±3.56;6周时SS组24.40±2.41,IR组28.80±2.77,IR1d+HBO组51.00±4.30,IR7d+HBO组42.00±3.39,IR14d+HBO组34.80±2.58,IR28d+HBO组31.30±3.41;SVZ BrdU/Nestin阳性细胞数在同期各组间差异均有统计学意义(P<0.05);进一步两两比较显示,与SS组比较,除第8周,各时间点IR组大鼠SVZ BrdU/Nestin阳性细胞数均增多(P<0.05);各时间点IR1d+HBO组大鼠SVZ BrdU/Nestin阳性细胞数均多于IR组(P<0.05),在第2、4、6周,IR7d+HBO、IR14d+HBO、IR28d+HBO组大鼠SVZ BrdU/Nestin阳性细胞数均多于IR组(P<0.05);各组均在第2周/最初测量点时SVZ BrdU/Nestin阳性细胞数达到最多(P<0.05)。结论脑梗死后越早时间开始HBO治疗,越能促进SVZ、DG区内源性NSCs的增殖;脑梗死晚期,HBO治疗对内源性NSCs增殖已基本无影响。

Objective To observe the effects of hyperbaric oxygen(HBO)at different courses of treatment on the proliferation of endogenous neural stem cells(NSCs)in cerebral ventricular subventricular zone(SVZ)and hippocampal dentate gyrus(DG)in rats with focal cerebral ischemia at different periods.Methods The model of focal cerebral ischemia-reperfusion was performed by middle cerebral artery occlusion(MCAO).A total of 105 rats were divided by lottery into pseudo-surgery group(SS),ischemia-reperfusion group(IR group),HBO at 1st day after ischemia-reperfusion group(IR1d+HBO group),HBO at 7th day after ischemia-reperfusion group(IR7d+HBO group),HBO at 14th day after ischemia-reperfusion group(IR14d+HBO group),and HBO at 28th day after ischemia-reperfusion group(IR28d+HBO).The SVZ and DG-derived NSCs were detected by immunofluorescence BrdU/Nestin double labeling at the 2nd,4th,6th,and 8th week after modeling.Results At the 2nd week after modeling,the number of DG BrdU/Nestin positive cells was 21.20±2.58 in the SS group,56.40±4.51 in the IR group,82.80±7.19 in the IR1d+HBO group,and 70.00±5.09 in the IR7d+HBO group.At the 4th week,it was 21.00±2.92 in the SS group,37.20±3.27 in the IR group,70.60±5.12 in the IR1d+HBO group,57.20±3.56 in the IR7d+HBO group,45.80±4.32 in the IR14d+HBO group.At the 6th week,it was 20.20±1.92 in the SS group,26.40±2.74 in the IR group,48.00±3.16 in the IR1d+HBO group,40.60±3.36 in the IR7d+HBO group,31.60±2.41 in the IR14d+HBO group,26.60±2.30 in the IR28d+HBO group.The number of DG BrdU/Nestin positive cells was statistically significant among all subgroups during the same period(P<0.01)except for that at the 8th week.The pairwise comparison showed the numbers of DG BrdU/Nestin positive cells in the IR group at all time points were all higher than those in the SS group at the same time point(P<0.05).Except for that at the 8th week,all the numbers of DG BrdU/Nestin positive cells in the IR1d+HBO group,the IR7d+HBO group,and the IR14d+HBO group at each time point were higher than those in the IR group(P<0.05).In all the subgroups,the number of DG BrdU/Nestin positive cells reached the highest point at the 2nd week/the initial measurement point(P<0.05).At the 2nd week after modeling,the number of SVZ BrdU/Nestin positive cells was 25.20±2.86 in the SS group,66.40±2.96 in the IR group,90.40±6.50 in the IR1d+HBO group,75.00±4.58 in the IR7d+HBO group.At the 4th week,it was 24.20±1.48 in the SS group,49.80±4.32 in the IR group,72.40±4.92 in the IR1d+HBO group,57.80±6.46 in the IR7d+HBO group,43.80±3.56 in the IR14d+HBO group.At the 6th week,it was 24.40±2.41 in the SS group,28.80±2.77 in the IR group,51.00±4.30 in the IR1d+HBO group,42.00±3.39 in the IR7d+HBO group,34.80±2.58 in the IR14d+HBO group,31.30±3.41 in the IR28d+HBO group.The number of SVZ BrdU/Nestin positive cells was statistically significant among all subgroups during the same period(P<0.05).The pairwise comparison showed the numbers of SVZ BrdU/Nestin positive cells in the IR group at all time points were all higher than those in the SS group at the same time point(P<0.05)except for the 8th week.The numbers of SVZ BrdU/Nestin positive cells in the IR1d+HBO group at each time point were higher than that in the IR group(P<0.05).The numbers of SVZ BrdU/Nestin positive cells in the IR7d+HBO group,the IR14d+HBO group,and the IR28d+HBO group at the 2nd,4th,and 6th week were higher than those in the IR group at the same time point(P<0.05).In all the subgroups,the number of SVZ BrdU/Nestin positive cells reached the highest point at the 2nd week/the initial measurement point(P<0.05).Conclusion The earlier HBO treatment on cerebral infarction starts,the better promotion of the proliferation of SVZ and DG-derived NSCs achieves.HBO treatment has almost no effect on the proliferation of endogenous NSCs at late cerebral infarction.