慢性缺氧对新生大鼠脑白质及脑发育的影响

Impact of chronic hypoxia on white matter and brain development in neonatal rat model

目的研究慢性缺氧对新生大鼠脑白质(white matter,WM)损伤及脑发育延迟的作用,探讨该模型与紫绀型先天性心脏病(congenital heart defects,CHD)慢性缺氧脑损伤的相似性。方法选取3日龄(P3)新生Sprague-Dawley大鼠随机分为实验组[n=36,吸入氧浓度(FiO2)为10.5%±1.0%]和对照组(n=36,FiO2为21.0%±0.0%),饲养12 d至P14。检测:(1)每日测量SD大鼠体重变化及P14新鲜脑重;(2)P14脑切片H&E染色:观察脑组织病理变化;(3) P14脑切片免疫组织化学染色:观察脑WM区域少突胶质祖细胞(oligodendroglial progenitor cells,OPC)、少突胶质前体细胞(preoligodendrocytes,PreOL)和髓磷脂蛋白(myelin basic protein,MBP)变化;(4)P14新鲜鼠脑WM区域约50 mg,组织蛋白抽提,Western blotting方法定量分析MBP表达量变化;(5)行为学测试:每组各留9只饲养至P30,行转棒实验评估运动功能和协调能力。结果 (1)实验组体重、脑重增长明显慢于对照组(P14)[体重(14.92±1.26)g vs.(30.26±1.81)g,t=7.51,P<0.01;脑重(0.68±0.05) g vs.(0.97±0.04)g,t=13.26,P<0.01];(2)HE染色:实验组脑室扩大(P<0.01),脑WM区域水肿、结构疏松杂乱,局部软化坏死灶,细胞排列紊乱;(3)免疫组织化学染色:实验组OPC、PreOL数量少于对照组(64.8±6.3 vs. 126.2±8.4,t=11.19,P<0.01;19.1±7.6 vs. 46.7±9.5,t=7.28,P<0.01),实验组MBP比对照组稀疏、杂乱;(4)Western blotting检测:实验组MBP较对照组表达量明显下降(P<0.01);(5)行为学测试:实验组棒上停留时间较对照组明显缩短(P<0.01)。结论慢性缺氧可造成新生大鼠脑WM损伤和脑发育延迟,具有紫绀型CHD围产期慢性缺氧WM损伤和脑发育不成熟的相似特征。

Objective To study the impact of chronic hypoxia on white matter（WM） injury and brain development delay using a neonatal rat model, and to explore its value in simulating chronic hypoxic brain damage in cyanotic congenital heart disease（CHD）. Methods Three-day-old Sprague-Dawley（SD） rats were randomly distributed to an experiment group（n=36, FiO2 10.5%±1.0%） and a control group（n=36, FiO2 21.0%±0.0%） and were raised for 12 days.（1） Body weight of SD rats was recorded every day and fresh brain weight was measured on P14.（2） HE staining was performed on sections of brain tissue to observe pathological changes and ventricular size.（3） Immunohistochemistry（IHC） was applied to reveal alterations of oligodendroglial progenitor cells（OPC）, preoligodendrocytes（PreOL） and myelin basic protein（MBP） in brain WM area.（4） Protein was extracted from 50 mg of brain tissue in WM area and expression of MBP was determined using Western blotting.（5） Motor function and coordination of rats（P30） were assessed via rotation experiment. Results（1） Body weight and brain weight were significantly less in the experiment group compared with the control group on P14（body weight 14.92±1.26 g vs. 30.26±1.81 g, t=7.51, P〈0.01; brain weight 0.68±0.05 g vs. 0.97±0.04 g, t=13.26, P〈0.01）;（2） HE staining： Sections of brain tissue from the experiment group showed ventricular size enlargement with a statistical difference（P〈0.01）, disordered cell organization, local neuronal death and leukomalacia.（3） The number of OPC and PreOL in the experiment group were significantly less than those in the control group（64.8±6.3 vs. 126.2±8.4,t=11.19, P0.01; 19.1±7.6 vs. 46.7±9.5, t=7.28, P〈0.01, respectively）. MBP distribution was sparse and disorganized in the experiment group.（4） Western blotting： Expression of MBP was less in the experiment group（P〈0.01）.（5） Behavioral test： Time on rotarod was less in the experiment group with a statistical difference（P〈0.01）.Conclusion Chronic hypoxia can result in WM injury and brain development delay in neonatal rats, with features comparable to those seen in infants with cyanotic CHD.