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目的研究血管性痴呆大鼠皮层和海马区神经细胞线粒体氧化应激的病理学变化及其意义。方法 Wistar大鼠按照体重随机分为假手术组和模型组,结扎双侧颈总动脉建立血管痴呆模型,术后3个月,用八臂迷宫和新物体识别观察大鼠学习记忆能力。大鼠实施安乐死,提取分离线粒体,用分光光度法测量线粒体肿胀作为膜孔通透性指标,用多功能酶标仪分别测量JC-1、DCFH-DA、DHE和DHR123荧光强度变化作为线粒体膜电位、线粒体内总氧氮自由基(ROS/RNS)、超氧阴离子(O_2~-)和过氧亚硝基阴离子(ONOO~-)的变化指标。结果模型组的大鼠学习记忆错误次数为(3.57±1.13),显著高于假手术组的(2.00±0.82;P<0.05);新物体识别,模型组和假手术组辨别指数分别为-0.02±0.04和0.21±0.02,辨别潜伏期分别为(23.87±2.99),(10.35±4.67)s,模型组潜伏期显著延长(P<0.05)。与假手术组比较,模型组大鼠皮层和海马神经细胞线粒体膜电位显著下降、膜孔通透性增加、ROS/RNS、O_2~-和ONOO~-含量均显著上升(P<0.0001)。结论血管痴呆大鼠皮层和海马区神经细胞线粒体氧化应激水平明显增高,可能是血管痴呆大鼠认知障碍的主要机制之一。
Objective To study the pathological changes of mitochondrial oxidative stress in the cortex and hippocampus of vascular dementia rats and its significance. Methods Wistar rats were randomly divided into sham operation group and model group according to body weight. The bilateral common carotid arteries were ligated to establish vascular dementia model. Three months after operation, the learning and memory abilities of rats were observed by eight-armed maze and new object recognition. The rats were euthanized and the mitochondria were extracted and separated. The mitochondrial swelling was measured by spectrophotometry as the index of membrane pore permeability. Fluorescence intensity changes of JC-1, DCFH-DA, DHE and DHR123 were measured by multifunctional microplate reader as mitochondrial membrane potential (ROS / RNS), superoxide anion (O_2 ~ -) and peroxynitrite anion (ONOO ~ -) in mitochondria. Results The number of learning and memory errors in the model group was (3.57 ± 1.13), significantly higher than that in the sham-operated group (2.00 ± 0.82; P <0.05). The discrimination index of new object recognition model group and sham operation group was -0.02 ± 0.04 and 0.21 ± 0.02, respectively. The latent periods were (23.87 ± 2.99) and (10.35 ± 4.67) s, respectively. The latency of model group was significantly longer (P <0.05). Compared with the sham operation group, the mitochondrial membrane potential of cortical and hippocampal neurons in model group decreased significantly, and the permeability of membrane pores increased. The content of ROS / RNS, O_2 ~ - and ONOO ~ - increased significantly (P <0.0001). Conclusion The mitochondrial oxidative stress in the cortex and hippocampus of vascular dementia rats is significantly increased, which may be one of the main mechanisms of cognitive impairment in vascular dementia rats.