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目的采用双侧颈总动脉结扎法(2VO)建立慢性脑缺血性模型,研究藁本内酯(LIG)对脑白质损伤的保护作用。方法 48只雄性Wistar大鼠,随机分为假手术组、模型组、LIG40和10 mg·kg-1.d-1),2VO建立大鼠脑血流低灌的缺血性模型,术后1 h灌胃给药。(1)伊文思蓝(EB)扩散试验:每组6只动物,连续给药3 d,末次药后1 h,静脉注射4%伊文思蓝(1 ml/只),1 h后麻醉动物,PBS行心脏快速灌流后开颅取脑,以液氮速冻,制备连续冠状冰冻切片(20μm),于荧光显微镜G激发状态下观察,NIH Image1.6图像分析软件分析EB渗漏,以比较各组血脑屏障通透性的变化;(2)对慢性缺血性脑白质损伤的影响:每组6只动物,连续给药14d,末次药后1 h,麻醉动物取脑、常规固定,用Klüver-Barrer(KB)染色观察胼胝体、内囊、尾壳核髓鞘损伤程度,免疫组织化学染色观察胼胝体区域髓鞘碱性磷脂蛋白(MBP)、胶质纤维酸性蛋白(GFAP)和基质金属蛋白酶-2(MMP-2)的蛋白表达,Image-Pro-Plus 5.0图像分析软件计算结果。结果与假手术组比较,模型组脑内的EB渗出量显著增加(P<0.01);KB染色显示胼胝体、内囊、尾壳核白质组织出现明显空洞和神经纤维脱失,胼胝体MBP表达显著减少;胼胝体GFAP及MMP-2表达显著增加(P<0.01);而LIG剂量依赖性显著减少2VO大鼠脑内的EB渗出量、改善髓鞘脱失和与空泡形成等白质疏松程度、抑制胼胝体GFAP和MMP-2的蛋白表达(P<0.05)。结论 LIG可明显减轻慢性缺血性脑白质损伤,其机制可能与LIG可改善缺血急性期血脑屏障功能、抑制胶质细胞活化以及基质金属蛋白酶活性有关。
Objective To establish a chronic cerebral ischemic model by bilateral common carotid artery ligation (2VO) to study the protective effect of ligustilide (LIG) on white matter damage. Methods Forty-eight male Wistar rats were randomly divided into sham-operation group, model group, LIG40 and 10 mg · kg-1.d-1, and 2VO was used to establish ischemic model of cerebral hypoperfusion in rats. h gavage. (1) Evans blue (EB) diffusion test: Six animals in each group were given continuous administration for 3 days. IV Evans Blue (1 ml / body) was injected intravenously 1 h after the last treatment. Animals were anesthetized 1 h later, The hearts of rats were perfused with PBS immediately after cardioplegia, then frozen in liquid nitrogen to prepare continuous coronal frozen sections (20μm). The cells were observed under fluorescence microscope G excited state. NIH Image1.6 image analysis software was used to analyze the EB leakage to compare the different groups Blood brain barrier permeability changes; (2) the impact of chronic ischemic white matter damage: six animals in each group, continuous administration of 14d, 1h after the last drug, anesthetized animals were brains, routine fixation, with Klüver The degree of myelin damage of the corpus callosum, the inner capsule and the caudate putamen were observed under a light-barreled (KB) staining. The levels of MBP, GFAP and MMP-9 in the corpus callosum were observed by immunohistochemical staining. 2 (MMP-2) protein expression, Image-Pro-Plus 5.0 image analysis software to calculate the results. Results Compared with sham operation group, EB exudation in model group increased significantly (P <0.01); KB staining showed obvious vacuoles and nerve fiber loss in white matter of corpus callosum, inner capsule and caudate putamen, and significant expression of MBP in corpus callosum (P <0.01). However, LIG dose-dependently decreased the amount of EB exudation in the brain of 2VO rats and the degree of white matter loss, such as demyelination and vacuolization, in the corpus callosum, Inhibit the protein expression of corpus callosum GFAP and MMP-2 (P <0.05). Conclusion LIG can significantly reduce chronic ischemic white matter injury. The possible mechanism is that LIG can improve the function of blood-brain barrier and inhibit the activation of glial cells and the activity of matrix metalloproteinase in acute phase of ischemia.