摘要
目的观察缺氧缺血(HI)新生大鼠脑白质损伤时少突胶质细胞(OLs)的作用。方法将48只出生3 d的清洁级SD大鼠分为实验组和对照组。实验组大鼠分离右侧颈总动脉、结扎离断,缺氧处理;对照组大鼠分离右侧颈总动脉,不结扎离断,也不进行缺氧处理。术后3 d、7 d、14 d心脏灌注后取其脑组织,各时间点实验组与对照组分别标记为A1/A2,B1/B2,C1/C2。处死大鼠的前24 h腹腔注射5-溴脱氧尿嘧啶核苷(Brdu),每4 h 1次,连续注射3次。脑组织经固定、脱水处理后冷冻切片。对脑组织切片进行焦油紫(CV)染色,评估脑白质损伤情况。行神经元-神经胶质2型抗原(NG-2)、髓鞘碱性蛋白(MBP)、Brdu、含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)的免疫组织化学染色,观察缺氧缺血性脑损伤新生大鼠OLs的作用。结果 1.CV染色显示脑面积比率:A1组损伤侧与损伤对侧脑面积比率为0.900±0.059、A2组左侧与右侧脑面积比率为0.970±0.015、B1组损伤侧与损伤对侧脑面积比率为0.870±0.039、B2组左侧与右侧脑面积比率为0.920±0.024、C1组损伤侧与损伤对侧脑面积比率为0.730±0.096、C2组左侧与右侧脑面积比率为0.920±0.039,实验组和对照组间比较差异有统计学意义(P<0.05)。胼胝体面积比率:A1组损伤侧与损伤对侧胼胝体面积比率(0.870±0.028)和A2组左侧与右侧胼胝体面积比率(0.910±0.033)比较差异有统计学意义(P=0.049)。B1组损伤侧与损伤对侧胼胝体面积比率(0.92±0.030)和B2组左侧与右侧胼胝体面积比率(0.92±0.025),C1组损伤侧与损伤对侧胼胝体面积比率(0.83±0.130)和C2组左侧与右侧胼胝体面积比率(0.85±0.076)比较,差异均无统计学意义(P>0.05)。2.MBP染色:阳性面积占整个胼胝体的比率:B1组损伤侧和损伤对侧为0.32±0.17、0.39±0.20;B2组左右两侧阳性面积占整个胼胝体面积比率为0.51±0.11;C1组损伤侧和损伤对侧为0.71±0.07、0.90±0.10;C2组左右两侧阳性面积占整个胼胝体面积比率为0.88±0.13,各个时间点实验组损伤侧和损伤对侧、实验组和对照组间比较差异均有统计学意义(Pa<0.05)。3.NG-2染色:每高倍镜下阳性细胞数:A1组损伤侧和损伤对侧为23.56±2.93、18.11±3.19;A2组左右两侧20.17±2.38;B1组损伤侧和损伤对侧为27.29±4.81、22.88±5.31;B2组左右两侧20.81±3.24;C1组损伤侧和损伤对侧为22.29±4.27、16.04±4.17;C2组左右两侧14.07±3.29。各个时间点实验组损伤侧和损伤对侧、实验组和对照组间比较差异均有统计学意义(P<0.05)。4.Caspase-3染色:每高倍镜下阳性细胞数:A1组损伤侧和损伤对侧为68.67±6.60、60.04±5.66;A2组两侧40.21±7.93;B1组损伤侧和损伤对侧为59.13±10.22、50.38±11.58;B2组两侧为48.17±4.27;C1组损伤侧和损伤对侧为70.13±16.11、57.00±15.43;C2组两侧62.42±13.56。各个时间点实验组损伤侧和损伤对侧、术后3 d、7 d实验组和对照组间差异均有统计学意义(Pa<0.05)。5.Brdu染色:每高倍镜下阳性细胞数:A1组损伤侧和损伤对侧为149.58±21.12、126.92±7.23;A2组两侧为120.33±7.18;B1组损伤侧和损伤对侧为89.04±20.42、79.79±16.77;B2组两侧为85.83±6.05;C1组损伤侧和损伤对侧为60.08±10.89、53.25±8.02;C2组两侧为50.08±4.78。术后3 d、7 d实验组损伤侧和损伤对侧、实验组和对照组间比较差异均有统计学意义(Pa<0.05)。结论 HI可引起OLs成熟、髓鞘形成障碍,导致脑的低髓鞘化,从而引起脑白质损伤;OLs是缺氧缺血性脑损伤的靶细胞。
Objective To investigate the role of oligodendrocytcs (OLs) in the hypoxic - ischemic encephalopathy of immature rats. Methods Forty - eight 3 - days old SD rats were divided into 2 groups : the experimental group and the control group. In the experimental group,the right common carotid artery of the immature rats were isolated and cut off, then they were put in the hypoxic box. However, in the control group,the right common artery of the immature rats were only isolated without other treatment. The specimen of brain tissue was a- chieved on 3 days,7 days, and 14 days after the surgery with heart perfusion separately. They were labeled as group A 1/A2 ,B1/B2 , C1/C2. Be- fore the perfusion,the rats got 3 times of Brdu by intraperitoneal injection,every 4 hours once. Then the brain tissue was cut into slices after the fixing and dewatering. The brain section was dyed with Cresyl fast videt(CV) to evaluate the damage of brain. The immunohistochemistry staining of the brain section, targeting with neuron - glial antigen 2 ( NG - 2 ) , myelin basic protein (MBP) ,5 - Bromo - 2 - deoxyUridine ( Br-du) , and Caspase - 3 were performed. To investigate the role of OLS in the hypoxic - ischemic encephalopathy of immature rats. Results 1. There were significant differences between A1 group and A2 group (0. 900 ± 0. 059 vs O. 970± 0. 015 ) ; Bt group and B2 group (0. 870 ±0. 039 vs 0. 920 ± 0. 024 ) ; C1 group and C2 group (0. 730 ±0. 096 vs 0. 920 ±0. 039 ) in the ratio of brain area with the staining of CV ( P 〈 0.05 ). Meanwhile, there were significant differences between AI group and A2 group (0. 870 ± 0. 028 vs 0. 910 ± 0. 033 )in the ratio of corpus callosum. There were no significant differences between B1 group and B2 group(0. 920±0. 030 vs O. 920 +0. 025),C1 group and C2 group (0. 830 ± 0. 130 vs O. 850 ± 0. 076) ( P 〉 0.05 ). 2. The ratio of MBP positive area of the corpus callosum and the whole corpus callosum : which in the damage part and the contralateral part in B~ group were (0.32 ±0.17 ), (0.39 ± 0.20) ;in group B2 was 0.51 ± 0.11 ;which in the damage part and the c ontralateral part in C~ group were (0.71±0.07 ), (0.90 ± 0.10 ) ;in C2 group was 0.88 ± 0.13. There were signifi- cant differences between the damage part and the contralateral part at every time point ( Pa 〈 0.05 ) ; and there were also significant differences between the experimental group and the control group at every time point( P 〈 0.05 ). 3. The number of NG - 2 positive ceils at higher magni- fication:which in the damage part and the contralateral part in A1 group were (23.56 ± 2.93 ), (18.11 ± 3.19 ) ;in A2 group was (20. 17 ± 2.38) ;which in the damage part and the contralateral part in BI group were (27.29 ± 4.81 ), (22.88 ± 5.31 ) ; in B2 group was (20.81 -+ 3.24 ) ;which in the damage part and the contralateral part in C1 group were (22.29 ± 4.27), (16.04 ±4.17 ) ;in group C2 (14.07 ± 3.29). There were significant differences between the damage part and the contralateral part at every time point (Pa 〈 0. 05 ) ;and there were also sig- nificant differences between the experimental group and the control group at every time point (P 〈 0.05 ). 4. The number of Caspase -3 posi- tive cells at higher magnification:which in the damage part and the contralateral part in A1 group were (68.67 ± 6.60 ), (60.04 ± 5.66 ) ;in A2 group was 40.21 +7.93 ;which in the damage part and the contralateral part in B1 group were(59.13± 10.22), (50.38 ± 11.58) ;in B2 group was (48.17 ± 4.27 ) ;which in the damage part and the contralateral part in C1 group were (70.13 ±16.11 ), (57.00 ± 15.43 ) ;in C2 group was(62.42 ± 13.56). There were significant differences between the damage part and the contralateral part at every time point (P, 〈 0.05 ) ;and there were also significant differences between the experimental group and the control group at the first two time points (P〈 〈 0.05 ). 5. The number of Brdu positive cells at higher magnification : in the damage part and the contralateral part in A1 group were( 149.58 ± 21.12), (126.92 ± 7.23 );in A2 group was( 120.33 ± 7. 18 );in the damage part and the contralateral part in B1 group were (89.04 ± 20.42 ), (79.79 ±16.77 ) ;in B2 group was (85.83 ± 6.05 ) ;in the damage part and the contralateral part in C1 group were (60.08 ±10.89), ( 53.25 ± 8.02 ) ; in C2 group was( 50.08 ± 4.78 ). There were significant differences between the damage part and the contralateral part at the first two time points ( P 〈 0.05 ) ; and there were also significant differences between the experimental group and the control group at the first two time points( P 〈 0.05 ). Conclusions The hypoxic - ischemic injury can lead to a deficit in mature, myelin - producing OLs and as a consequence,cerebral hypomyelination;and OLs are the main targets of the injury.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2012年第22期1740-1744,共5页
Journal of Applied Clinical Pediatrics
基金
上海市卫生局科研计划课题资助项目(2009093)
卫生部新生儿疾病重点实验室开放基金课题(2012)
关键词
少突胶质细胞
缺氧缺血
脑白质损伤
未成熟脑
髓鞘化障碍
oligodendrocytes
hypoxia - ischemia
white matter injury
immature brain
dysfunction of myelination