本文選題：精神分裂癥 + 大腦皮質(zhì)��； 參考：《重慶醫(yī)科大學(xué)》2013年碩士論文

【摘要】：精神分裂癥是一種病因尚未完全闡明的精神疾病，以前認為精神分裂癥是一種功能性疾病，患者無器質(zhì)性改變。但是隨著科學(xué)的進步，越來越多的證據(jù)表明精神分裂癥存在大腦結(jié)構(gòu)的改變。一些研究者觀察到精神分裂癥大腦皮質(zhì)存在區(qū)域性變薄，大腦皮質(zhì)神經(jīng)元密度增加，而大腦皮質(zhì)神經(jīng)元數(shù)量未發(fā)生顯著性改變。神經(jīng)影像學(xué)研究表明精神分裂癥大腦白質(zhì)纖維束的改變，皮質(zhì)與皮質(zhì)結(jié)構(gòu)完整性受到破壞，同時病理學(xué)研究結(jié)果表明大腦皮質(zhì)內(nèi)少突膠質(zhì)細胞腫脹和丟失，髓鞘結(jié)構(gòu)的破壞，基因微陣列分析顯示髓鞘相關(guān)基因明顯下調(diào)。雖然這些研究都觀察到了精神分裂癥大腦白質(zhì)、皮質(zhì)有髓神經(jīng)纖維存在顯著性脫髓鞘改變，并認為有髓神經(jīng)纖維脫髓鞘改變導(dǎo)致的大腦連接功能障礙在精神分裂癥的發(fā)病機制中起著重要作用，但是，到目前為止，國內(nèi)、外已有的研究均沒有對精神分裂癥大腦皮質(zhì)有髓神經(jīng)纖維及髓鞘進行準確的三維形態(tài)定量研究。本實驗中我們選用公認的CPZ（cuprizone，雙環(huán)己酮草酰二腙）動物模型探討精神分裂癥大腦皮質(zhì)有髓神經(jīng)纖維及髓鞘的改變，運用新的體視學(xué)方法和透射電子顯微鏡技術(shù)研究大腦皮質(zhì)有髓神經(jīng)纖維總長度、總體積、平均直徑以及髓鞘的總體積、內(nèi)外周長、厚度等的改變。 第一部分 1材料與方法 1.1實驗動物 6周齡雄性C57BL/6小鼠（重慶醫(yī)科大學(xué)實驗動物中心提供），分為CPZ組和對照組，CPZ組9只，用含0.2%CPZ的飼料飼養(yǎng)6周，對照組10只，用正常飼料飼養(yǎng)6周。每隔2～3天稱量一次動物體重。 1.2行為學(xué)實驗 CPZ組和對照組小鼠在第6周進行行為學(xué)實驗以檢測CPZ處理6周后小鼠的行為學(xué)改變，實驗項目包括曠場實驗、高架十字迷宮實驗、Morris水迷宮實驗、探孔實驗、轉(zhuǎn)棒實驗。 1.3行為學(xué)實驗結(jié)束后，對小鼠進行麻醉灌注固定，取出大腦，運用免疫組化染色定性觀察大腦皮質(zhì)脫髓鞘改變。 1.4隨機選擇一側(cè)大腦半球，作1mm連續(xù)等距腦組織切片，估算大腦皮質(zhì)體積，每組小鼠隨機選取5只動物送電鏡標本，每只動物抽取5塊組織塊，制作超薄切片，在透射電鏡下放大6000倍，隨機選取10個視野拍照，每只動物共拍攝50張照片。 1.5利用體視學(xué)方法計算CPZ組和對照組小鼠大腦皮質(zhì)體積，皮質(zhì)內(nèi)有髓神經(jīng)纖維總體積、總長度、平均直徑，并對有髓神經(jīng)纖維總長度進行分段統(tǒng)計。 2結(jié)果 2.1動物體重的變化情況：實驗開始后CPZ組小鼠體重在前兩周時間內(nèi)出現(xiàn)先急劇下降，后緩慢回升現(xiàn)象，對照組小鼠體重均顯著性高于CPZ組小鼠。 2.2行為學(xué)實驗結(jié)果： 與對照組小鼠相比，CPZ組小鼠在曠場實驗中中央?yún)^(qū)域活動增多，高架十字迷宮實驗中開臂內(nèi)活動增多，提示CPZ組小鼠焦慮行為減少，在水迷宮實驗、探孔實驗和轉(zhuǎn)棒實驗中兩組小鼠行為學(xué)表現(xiàn)無顯著性差異。 2.3免疫組化染色定性研究： CPZ組小鼠較對照組小鼠大腦皮質(zhì)出現(xiàn)明顯的脫髓鞘改變。 2.4體視學(xué)結(jié)果： CPZ組小鼠與對照組小鼠大腦皮質(zhì)總體積沒有顯著性差異（P0.05），CPZ組小鼠大腦皮質(zhì)有髓神經(jīng)纖維總體積比對照組小鼠顯著性降低了69.8%（P 0.01）。CPZ組小鼠大腦皮質(zhì)有髓神經(jīng)纖維總長度比對照組小鼠顯著性降低了68.9%（P 0.01），，直徑為0.2～＜0.4μm、0.4～＜0.6μm和0.6～＜0.8μm的大腦皮質(zhì)有髓神經(jīng)纖維總長度與對照組小鼠相比分別顯著性減少了4.317km、3.313km和0.940km（P0.01），CPZ組小鼠大腦皮質(zhì)有髓神經(jīng)纖維平均直徑顯著性增加了17.8%（P 0.01）。 第二部分 1材料與方法 1.1動物同第一部分 1.2電鏡標本同第一部分，在透射電鏡下放大17500倍后隨機選取15～20個視野拍照，每只動物平均拍攝90張電鏡照片。在電鏡照片下觀察有髓神經(jīng)纖維斷面髓鞘超微結(jié)構(gòu)。 1.3利用體視學(xué)方法計算CPZ組與對照組小鼠大腦皮質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘總體積、髓鞘厚度、髓鞘內(nèi)外周長，并計算不同直徑段的髓鞘厚度、髓鞘內(nèi)外周長差、髓鞘內(nèi)外周長比。 2結(jié)果 2.1電鏡觀察結(jié)果：與對照組相比，CPZ組小鼠大腦皮質(zhì)有髓神經(jīng)纖維存在明顯的脫髓鞘改變，可見明顯的髓鞘板層分離現(xiàn)象、髓鞘與軸突分離現(xiàn)象以及空泡樣改變。 2.2體視學(xué)研究結(jié)果 與對照組小鼠相比，CPZ組小鼠大腦皮質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘總體積顯著性減少了44%（P0.05），髓鞘平均厚度顯著性增加了31.5%（P0.01），兩組小鼠大腦皮質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘平均外周長不存在顯著性差異，但髓鞘平均內(nèi)周長存在顯著性差異趨勢（P=0.054），CPZ組小鼠大腦皮質(zhì)有髓神經(jīng)纖維髓鞘平均內(nèi)外周長之差較對照組顯著性增加了0.249μm（P0.01），CPZ組髓鞘內(nèi)外周長之比較對照組顯著性降低了11.6%（P0.01）。 與對照組小鼠相比，CPZ組小鼠髓鞘厚度在0.8μm以下（0.2～＜0.3μm除外）均顯著性增加，髓鞘外周長在0.2～＜0.3μm，0.8～＜0.9μm直徑段均顯著性降低，髓鞘內(nèi)周長在0～＜0.3μm，0.8～＜1.0μm直徑段均顯著性降低，髓鞘內(nèi)外周長之差在0～＜0.2μm，0.4～＜0.5μm，0.6～＜0.7μm直徑段顯著性增加，髓鞘內(nèi)外周長之差在≥1.0μm直徑段顯著性降低，髓鞘內(nèi)外周長之比在0.7μm以下（0.3～＜0.4μm除外）均顯著性降低，髓鞘內(nèi)外周長之比≥1.0μm直徑段顯著性增加（P0.05）。 全文結(jié)論 1. CPZ模型小鼠可以出現(xiàn)類似精神分裂癥樣癥狀的行為學(xué)改變。 2.獲得了CPZ組和對照組小鼠大腦皮質(zhì)體積，大腦皮質(zhì)有髓神經(jīng)纖維總體積、總長度、平均直徑以及大腦皮質(zhì)有髓神經(jīng)纖維髓鞘的總體積、髓鞘厚度、髓鞘內(nèi)外周長等基本數(shù)據(jù)，為今后運用該動物模型進行的其他研究提供了重要的參考數(shù)據(jù)。 3.發(fā)現(xiàn)CPZ組小鼠大腦皮質(zhì)有髓神經(jīng)纖維總體積、總長度顯著性降低，大腦皮質(zhì)有髓神經(jīng)纖維總長度的降低主要是由于小直徑纖維丟失造成的。 4.發(fā)現(xiàn)CPZ組小鼠大腦皮質(zhì)有髓神經(jīng)纖維髓鞘的丟失，為精神分裂癥的脫髓鞘改變假說提供了直接、定量的科學(xué)依據(jù)。
[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.
【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2013
【分類號】：R749.3

【參考文獻】

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本文編號：1965209