CPZ诱导的精神分裂症样小鼠大脑皮质体积、皮质内有髓神经纤维及髓鞘改变的体视学研究

发布时间：2018-06-01 18:32

本文选题：精神分裂症 + 大脑皮质　；参考：《重庆医科大学》2013年硕士论文

【摘要】：精神分裂症是一种病因尚未完全阐明的精神疾病，以前认为精神分裂症是一种功能性疾病，患者无器质性改变。但是随着科学的进步，越来越多的证据表明精神分裂症存在大脑结构的改变。一些研究者观察到精神分裂症大脑皮质存在区域性变薄，大脑皮质神经元密度增加，而大脑皮质神经元数量未发生显著性改变。神经影像学研究表明精神分裂症大脑白质纤维束的改变，皮质与皮质结构完整性受到破坏，同时病理学研究结果表明大脑皮质内少突胶质细胞肿胀和丢失，髓鞘结构的破坏，基因微阵列分析显示髓鞘相关基因明显下调。虽然这些研究都观察到了精神分裂症大脑白质、皮质有髓神经纤维存在显著性脱髓鞘改变，并认为有髓神经纤维脱髓鞘改变导致的大脑连接功能障碍在精神分裂症的发病机制中起着重要作用，但是，到目前为止，国内、外已有的研究均没有对精神分裂症大脑皮质有髓神经纤维及髓鞘进行准确的三维形态定量研究。本实验中我们选用公认的CPZ（cuprizone，双环己酮草酰二腙）动物模型探讨精神分裂症大脑皮质有髓神经纤维及髓鞘的改变，运用新的体视学方法和透射电子显微镜技术研究大脑皮质有髓神经纤维总长度、总体积、平均直径以及髓鞘的总体积、内外周长、厚度等的改变。第一部分 1材料与方法 1.1实验动物 6周龄雄性C57BL/6小鼠（重庆医科大学实验动物中心提供），分为CPZ组和对照组，CPZ组9只，用含0.2%CPZ的饲料饲养6周，对照组10只，用正常饲料饲养6周。每隔2～3天称量一次动物体重。 1.2行为学实验 CPZ组和对照组小鼠在第6周进行行为学实验以检测CPZ处理6周后小鼠的行为学改变，实验项目包括旷场实验、高架十字迷宫实验、Morris水迷宫实验、探孔实验、转棒实验。 1.3行为学实验结束后，对小鼠进行麻醉灌注固定，取出大脑，运用免疫组化染色定性观察大脑皮质脱髓鞘改变。 1.4随机选择一侧大脑半球，作1mm连续等距脑组织切片，估算大脑皮质体积，每组小鼠随机选取5只动物送电镜标本，每只动物抽取5块组织块，制作超薄切片，在透射电镜下放大6000倍，随机选取10个视野拍照，每只动物共拍摄50张照片。 1.5利用体视学方法计算CPZ组和对照组小鼠大脑皮质体积，皮质内有髓神经纤维总体积、总长度、平均直径，并对有髓神经纤维总长度进行分段统计。 2结果 2.1动物体重的变化情况：实验开始后CPZ组小鼠体重在前两周时间内出现先急剧下降，后缓慢回升现象，对照组小鼠体重均显著性高于CPZ组小鼠。 2.2行为学实验结果：与对照组小鼠相比，CPZ组小鼠在旷场实验中中央区域活动增多，高架十字迷宫实验中开臂内活动增多，提示CPZ组小鼠焦虑行为减少，在水迷宫实验、探孔实验和转棒实验中两组小鼠行为学表现无显著性差异。 2.3免疫组化染色定性研究： CPZ组小鼠较对照组小鼠大脑皮质出现明显的脱髓鞘改变。 2.4体视学结果： CPZ组小鼠与对照组小鼠大脑皮质总体积没有显著性差异（P0.05），CPZ组小鼠大脑皮质有髓神经纤维总体积比对照组小鼠显著性降低了69.8%（P 0.01）。CPZ组小鼠大脑皮质有髓神经纤维总长度比对照组小鼠显著性降低了68.9%（P 0.01），，直径为0.2～＜0.4μm、0.4～＜0.6μm和0.6～＜0.8μm的大脑皮质有髓神经纤维总长度与对照组小鼠相比分别显著性减少了4.317km、3.313km和0.940km（P0.01），CPZ组小鼠大脑皮质有髓神经纤维平均直径显著性增加了17.8%（P 0.01）。第二部分 1材料与方法 1.1动物同第一部分 1.2电镜标本同第一部分，在透射电镜下放大17500倍后随机选取15～20个视野拍照，每只动物平均拍摄90张电镜照片。在电镜照片下观察有髓神经纤维断面髓鞘超微结构。 1.3利用体视学方法计算CPZ组与对照组小鼠大脑皮质内有髓神经纤维髓鞘总体积、髓鞘厚度、髓鞘内外周长，并计算不同直径段的髓鞘厚度、髓鞘内外周长差、髓鞘内外周长比。 2结果 2.1电镜观察结果：与对照组相比，CPZ组小鼠大脑皮质有髓神经纤维存在明显的脱髓鞘改变，可见明显的髓鞘板层分离现象、髓鞘与轴突分离现象以及空泡样改变。 2.2体视学研究结果与对照组小鼠相比，CPZ组小鼠大脑皮质内有髓神经纤维髓鞘总体积显著性减少了44%（P0.05），髓鞘平均厚度显著性增加了31.5%（P0.01），两组小鼠大脑皮质内有髓神经纤维髓鞘平均外周长不存在显著性差异，但髓鞘平均内周长存在显著性差异趋势（P=0.054），CPZ组小鼠大脑皮质有髓神经纤维髓鞘平均内外周长之差较对照组显著性增加了0.249μm（P0.01），CPZ组髓鞘内外周长之比较对照组显著性降低了11.6%（P0.01）。与对照组小鼠相比，CPZ组小鼠髓鞘厚度在0.8μm以下（0.2～＜0.3μm除外）均显著性增加，髓鞘外周长在0.2～＜0.3μm，0.8～＜0.9μm直径段均显著性降低，髓鞘内周长在0～＜0.3μm，0.8～＜1.0μm直径段均显著性降低，髓鞘内外周长之差在0～＜0.2μm，0.4～＜0.5μm，0.6～＜0.7μm直径段显著性增加，髓鞘内外周长之差在≥1.0μm直径段显著性降低，髓鞘内外周长之比在0.7μm以下（0.3～＜0.4μm除外）均显著性降低，髓鞘内外周长之比≥1.0μm直径段显著性增加（P0.05）。全文结论 1. CPZ模型小鼠可以出现类似精神分裂症样症状的行为学改变。 2.获得了CPZ组和对照组小鼠大脑皮质体积，大脑皮质有髓神经纤维总体积、总长度、平均直径以及大脑皮质有髓神经纤维髓鞘的总体积、髓鞘厚度、髓鞘内外周长等基本数据，为今后运用该动物模型进行的其他研究提供了重要的参考数据。 3.发现CPZ组小鼠大脑皮质有髓神经纤维总体积、总长度显著性降低，大脑皮质有髓神经纤维总长度的降低主要是由于小直径纤维丢失造成的。 4.发现CPZ组小鼠大脑皮质有髓神经纤维髓鞘的丢失，为精神分裂症的脱髓鞘改变假说提供了直接、定量的科学依据。
[Abstract]:Schizophrenia is a mental disorder that has not yet been fully elucidated. Schizophrenia was previously considered a functional disease with no organic change in patients. But with the progress of science, more and more evidence suggests that schizophrenia has a structural change in the brain. Some researchers have observed the region of the cerebral cortex in schizophrenia. The density of the cortical neurons in the cerebral cortex was increased and the number of neurons in the cerebral cortex did not change significantly. The neuroimaging study showed the changes in the white matter fibers in the brain of schizophrenia, the damage of the cortical and cortical integrity, and the pathological results showed that the oligodendrocytes in the cerebral cortex were swollen and lost. The destruction of myelin structure and gene microarray analysis showed that the myelin related genes were obviously down-regulated. Although these studies have observed the white matter in the brain of schizophrenia, the myelinated nerve fibers in the cortex have significant demyelination, and the myelinated nerve fiber demyelinating changes in the brain connection dysfunction in the onset of schizophrenia. The mechanism plays an important role, but so far, there has been no accurate three-dimensional quantitative study on the myelinated nerve fibers and myelin sheath in the cerebral cortex of schizophrenia. In this experiment, we chose the accepted model of CPZ (cuprizone, dicyclohexanoyl two hydrazone) to explore the cerebral cortex of schizophrenia. Changes in myelinated nerve fibers and myelin sheath were used to study the total length, total volume, average diameter, total volume of myelin sheath, internal and external circumference and thickness of myelinated nerve fibers in the cerebral cortex using a new stereological method and transmission electron microscopy.
Part one
1 materials and methods
1.1 experimental animals
6 weeks old male C57BL/6 mice (provided by experimental animal center of Medical University Of Chongqing) were divided into CPZ group and control group, and 9 in group CPZ were fed with 0.2%CPZ feed for 6 weeks, and 10 in control group were fed with normal feed for 6 weeks. The animal weight was weighed every 2~3 days.
1.2 behavioral experiment
In group CPZ and control group, the behavior study was carried out at sixth weeks to test the behavioral changes of mice after 6 weeks of treatment. The experimental items included open field experiment, elevated cross maze test, Morris water maze experiment, hole exploration experiment, and rotating bar experiment.
1.3 after behavioral experiments, the mice were anesthetized and fixed, and the brain was removed. Immunohistochemical staining was used to observe the demyelination of cerebral cortex.
1.4 the cerebral hemispheres were randomly selected to slice the brain tissue of 1mm. The volume of cerebral cortex was estimated. 5 animals in each group were randomly selected for electron microscopy. 5 pieces of tissue were selected for each animal. The ultrathin sections were made, 6000 times magnified under transmission electron microscope, and 10 fields were photographed randomly. 50 photographs were taken for each animal.
1.5 the volume of cerebral cortex in group CPZ and control group was calculated by stereology. The total volume, total length and average diameter of the myelinated nerve fibers in the cortex were measured in the cortex, and the total length of the myelinated nerve fibers was statistically analyzed.
2 Results
2.1 the change of body weight: after the experiment, the body weight of group CPZ mice decreased rapidly in the first two weeks, then slowly recovered, and the weight of the control group was significantly higher than that in the CPZ group.
2.2 experimental results of behavioral studies:
Compared with the control group, the activity of central region in the CPZ group increased in the open field experiment, and the activity in the open arm increased in the elevated cross labyrinth experiment, suggesting that the anxiety behavior of the CPZ mice decreased. There was no significant difference between the two groups of mice in the water maze test and the rotation bar experiment.
2.3 immunohistochemical staining qualitative study: the CPZ group showed obvious demyelination in the cerebral cortex compared with the control group.
2.4 the results of stereology:
There was no significant difference in total volume of cerebral cortex between the CPZ group and the control group (P0.05). The total volume of the myelinated nerve fibers in the cerebral cortex of the CPZ group was 69.8% lower than that of the control group (P 0.01). The total length of the myelinated nerve fibers in the cerebral cortex of the.CPZ group was 68.9% (P 0.01) significantly lower than the control group, and the diameter was 0.2 ~ 0.2. The total length of myelinated nerve fibers in cerebral cortex of 0.4 mu m, 0.4 to 0.6 mu m and 0.6 ~ < 0.8 micron m significantly decreased 4.317km, 3.313km and 0.940km (P0.01), and the average diameter of myelinated nerve fibers in the cerebral cortex of CPZ mice increased by 17.8% (P 0.01).
The second part
1 materials and methods
1.1 animals and the first part
1.2 electron microscope specimens and the first part, 17500 times magnified under transmission electron microscope, randomly selected 15~20 fields of vision, each animal was photographed with an average of 90 electron microscopy photographs. The ultrastructure of myelin myelin sheath in the myelinated nerve fiber section was observed under the electron microscope.
1.3 the total medullary sheath volume, the thickness of myelin sheath and the circumference of myelin sheath in the cerebral cortex of the CPZ group and the control group were calculated by the stereological method, and the thickness of myelin sheath in different diameter segments, the difference in the circumference of the medullary sheath and the ratio of the circumference of the medullary sheath to the circumference of the myelin sheath were calculated.
2 Results
2.1 the results of electron microscopy: compared with the control group, there were obvious demyelinating changes in the medullary nerve fibers in the cerebral cortex of the CPZ group, and the separation of myelin sheath, the separation of myelin and axon and the change of vacuoles were obvious.
2.2 stereological findings
Compared with the control group, the total medullary sheath volume of myelinated nerve fibers in the cerebral cortex of the CPZ group was significantly reduced by 44% (P0.05), and the average thickness of myelin sheath was significantly increased by 31.5% (P0.01). The average peripheral length of myelin myelin sheath in the two groups of cerebral cortex of the two mice did not exist significant difference, but there was a significant difference in the mean perimeter of the myelin sheath. Trend (P=0.054), the difference in the mean and internal circumference between the medullary sheath of the myelinated nerve fibers in the cerebral cortex of the CPZ mice was 0.249 m (P0.01), and the comparison group in the CPZ group was 11.6% (P0.01).
Compared with the control group, the thickness of the myelin sheath in the CPZ group was significantly increased below 0.8 mu (0.2 ~ 0.3 mu m). The circumference of the myelin sheath was 0.2 to 0.3 mu m, and the 0.8 to 0.9 mu m diameter decreased significantly. The circumference of the myelin sheath was 0 to 0.3 u m, and the diameter of 0.8 ~ 1 m in the myelin sheath was significantly reduced, and the difference between the internal and external circumference of the myelin sheath was 0 ~ < 0.2 mu m, The diameter of 0.4 ~ < 0.5 m and 0.6 ~ 0.7 mu m increased significantly. The difference between the circumference of the myelin sheath and the diameter of the myelin sheath was significantly lower than that in the diameter of 1 mu m. The ratio of the circumference of the myelin sheath to the diameter of the myelin sheath was significantly lower than that of 0.7 m (0.3 to 0.4 mu m). The ratio of the circumference of the myelin sheath to the diameter of the myelin sheath was significantly increased (P0.05).
Full text conclusion
1. CPZ mice can exhibit behavioral changes similar to schizophrenia.
2. the volume of cerebral cortex in CPZ and control groups, the total volume of myelinated nerve fibers in the cerebral cortex, the total length, the average diameter, the total volume of myelin myelin sheath in the cerebral cortex, the thickness of myelin sheath and the circumference of the myelin sheath and other basic data were obtained, which provided important reference data for other studies in the future using the animal model.
3. the total volume of the medullary nerve fibers in the cerebral cortex of the CPZ group was found to be significantly reduced, and the decrease in the total length of the myelinated nerve fibers in the cerebral cortex was mainly caused by the loss of the small diameter fibers.
4. the loss of myelin sheath of myelinated nerve fibers in the cerebral cortex of CPZ mice was found, providing a direct and quantitative scientific basis for the hypothesis of demyelination in schizophrenia.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.3

【参考文献】