雌激素替代治疗对中老年卵巢切除大鼠大脑白质内有髓神经纤维及髓鞘超微结构作用的研究

发布时间：2018-01-25 04:14

本文关键词： 中老年卵巢切除大鼠白质髓鞘雌激素替代治疗体视学　出处：《重庆医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：体视学研究表明,老年大脑存在白质体积的显著性下降,并且主要是由于白质内有髓神经纤维的总长度下降引起的。并有研究表明，老年大脑存在认知功能的障碍，而女性在绝经后，雌激素的缺乏更会加速其认知功能下降的进程和增加其罹患阿尔茨海默氏病（Alzheimer’sdisease，AD）的风险。有髓神经纤维和髓鞘异常的结构是认知功能下降的重要原因。中枢神经系统作为雌激素的靶器官，已有众多的研究显示：雌激素可以改善大脑的认知能力和空间参考记忆能力。雌激素替代治疗能改善绝经后妇女包括白质在内的大脑结构。长期补充雌激素还能防护老年的白质丢失。可见，雌激素与大脑衰老和白质内有髓神经纤维及髓鞘之间的关系甚密。在本实验中，，我们对中老年雌性Sprague-Dawley（SD）大鼠行双侧卵巢切除术旨在模拟人类绝经后雌激素缺乏状态下大脑白质内有髓神经纤维及髓鞘结构异常的条件，并运用电镜技术及体视学三维精确定量方法研究雌激素替代治疗对卵巢切除大鼠白质及白质内有髓神经纤维髓鞘超微结构的作用，以了解雌激素对老年大脑白质和脱髓鞘病变的影响。本实验结果将为以后进一步运用雌激素替代治疗延缓大脑衰老进程和防治脱髓鞘疾病提供科学依据。第一部分1材料与方法 1.1中老年(10~12月龄)雌性SD大鼠40只(第三军医大学实验动物中心提供),在重庆医科大学实验动物中心饲养。将40只大鼠按体重以0.4ml/100g的浓度腹腔注射1%的戊巴比妥钠进行术前麻醉，然后行双侧卵巢切除术，术后连续3天对大鼠注射青霉素以抗感染。术后1月检测大鼠的血清雌激素水平。筛选出血清雌激素水平在11pg/ml左右的19只大鼠作为成功去势的卵巢切除模型。 1.2将筛选出的19只卵巢切除大鼠随机分为实验组与对照组（实验组10只，对照组9只），分别给予雌激素与安慰剂处理，4周后再次运用化学发光法检测血清雌激素水平。 1.3实验组与对照组大鼠在4周后进行Morris水迷宫行为学测试，以检测大鼠的空间参考记忆能力。 1.4Morris水迷宫测试结束后，从实验组随机抽取6只大鼠，从对照组随机抽取5只大鼠。然后对大鼠进行麻醉、灌注、固定，取出大脑。 1.5将左右大脑半球沿冠状线分别切成1mm厚的连续脑组织切片。每只大鼠随机抽取一侧大脑半球。为保证白质的各个部位都有相同的概率被抽取，在抽取的脑组织切片上随机叠放测点，在测点击中的地方抽取白质组织块，每只大鼠抽取5块电镜标本。切取1张60nm厚的超薄切片，放大20000倍后随机选取12个视野进行拍照，每只大鼠共拍得60张照片。 1.6运用体视学方法计算实验组和对照组大鼠白质总体积，白质内有髓神经纤维长度密度、总长度、体积密度、总体积、平均直径、轴突的平均直径、有髓神经纤维直径及其轴突直径之差、有髓神经纤维直径及其轴突直径之比，并对不同直径范围的白质内有髓神经纤维各统计参数进行比较。2结果 2.1雌激素水平的结果：术后检测的2组大鼠血清雌激素水平差异无统计学意义（P0.05）。雌激素替代治疗后检测的2组大鼠血清雌激素水平差异具有统计学意义（P 0.01）。 2.2Morris水迷宫测试结果：实验组大鼠的空间参考记忆能力较对照组显著性提高（P 0.05）。 2.3体视学结果：两组大鼠的白质总体积、白质内有髓神经纤维长度密度、总长度、体积密度、总体积、平均直径、轴突的平均直径、有髓神经纤维直径及其轴突直径之差、有髓神经纤维直径及其轴突直径之比均没有显著性差异（P0.05）。实验组大鼠在直径为1.2~1.4μm的大脑白质内有髓神经纤维总长度比对照组显著性增加了46.1%（P 0.05）。实验组大鼠在平均直径为0~0.4μm的大脑白质内有髓神经纤维直径及其轴突直径之差比对照组显著性增加了21.6%（P 0.05）。 1.材料与方法 1.1运用体视学方法计算实验组与对照组大鼠白质内有髓神经纤维髓鞘的体积密度、髓鞘的总体积、髓鞘的内周长、髓鞘的外周长、髓鞘的内外周长之差、髓鞘的内外周长之比、髓鞘的厚度。 1.2运用双变量相关分析统计学方法分析体视学参数和Morris水迷宫行为学参数之间的相关性。2.结果 2.1实验组大鼠大脑白质内有髓神经纤维髓鞘总体积比对照组显著性增加了27%(P 0.05)。实验组大鼠大脑白质内有髓神经纤维髓鞘内外周长之差比对照组显著性增加了12.6%（P 0.05）。 2.2实验组与对照组大鼠大脑白质内有髓神经纤维髓鞘的体积密度、内周长、外周长、内外周长之比、髓鞘的厚度均无统计学差异（P0.05）。 2.3实验组大鼠在平均直径为0~0.4μm的白质内有髓神经纤维直径及其轴突直径之差比对照组显著性增加了21.6%（P 0.05）。实验组大鼠在平均直径为0.4~0.6μm的白质内有髓神经纤维髓鞘内外周长之差比对照组显著性增加了20.7%（P 0.05），两组大鼠在平均直径为0.6~0.8μm的白质内有髓神经纤维髓鞘内外周长之差存在差异性趋势，实验组较对照组增加了14.3%（P=0.058）。 2.4白质内有髓神经纤维髓鞘总体积与Morris水迷宫第1天潜伏期及第4天潜伏期均呈负相关（P 0.05）。全文结论 1.雌激素替代治疗对中老年卵巢切除大鼠的空间参考记忆能力具有积极的保护作用。 2.获得了实验组和对照组大鼠大脑白质总体积，白质内有髓神经纤维长度密度、总长度、体积密度、总体积、平均直径、轴突的平均直径、有髓神经纤维直径及其轴突直径之差、有髓神经纤维直径及其轴突直径之比以及白质内有髓神经纤维髓鞘的总体积、内周长、外周长、内外周长之差、内外周长之比、髓鞘的厚度等基本数据，为以后进一步运用雌激素替代治疗延缓大脑衰老进程和防治脱髓鞘疾病提供了科学依据。 3.发现实验组大鼠大脑白质内有髓神经纤维髓鞘总体积、髓鞘的内外周长之差较对照组显著性增加。 4.发现与对照组相比，实验组大鼠在1.2~1.4μm直径段的白质内有髓神经纤维总长度显著性增加，在0~0.4μm直径段的白质内有髓神经纤维直径及其轴突之差显著性增加，在0.4~0.6μm直径段的髓鞘内外周长之差显著性增加，同时在0.6~0.8μm直径段的髓鞘内外周长之差存在差异性趋势。这些研究结果为实验组大鼠髓鞘总体积较对照组显著性增加提供了结构基础。 5.发现白质内有髓神经纤维髓鞘总体积与Morris水迷宫第1天潜伏期和第4天潜伏期具有相关性。
[Abstract]:A stereological study shows that elderly brain white matter volume was significantly decreased, and is mainly due to the total length of the myelinated fibers in the white matter decreased. And studies have shown that older brain cognitive function disorder, and in postmenopausal women, estrogen deficiency will accelerate the decline in cognitive function the process and increase the risk of Alzheimer's disease (Alzheimer sdisease, AD). The risk structure of myelinated nerve fibers and myelin abnormalities is an important cause of decline in cognitive function. The central nervous system as target organ of estrogen, has numerous studies show that the cognitive ability and spatial reference memory ability of estrogen can improve the brain estrogen replacement therapy can improve postmenopausal women, including the structure of white matter brain white matter. Long term estrogen can protective elderly lost. Obviously, estrogen and brain aging Old and in the white matter of the relationship between nerve fibers and myelin is very close. In this experiment, we in elderly female Sprague-Dawley (SD) rats underwent bilateral ovariectomy to simulate human postmenopausal estrogen deficiency of cerebral white matter in myelinated nerve fibers and myelin structure abnormal conditions, and the use of electron microscope technology and science of 3D precise quantitative stereological study of estrogen replacement therapy method of ovariectomized rat white matter and myelinated fibers in the white matter of the myelin sheath ultrastructure, in order to understand the estrogen in elderly cerebral white matter demyelination and influence. The experiment results for the further use of estrogen replacement therapy to delay the aging process of the brain and Prevention and treatment of demyelinating diseases and provide scientific basis.
The first part 1 materials and methods
1.1 in the aged (10~12 months) 40 female SD rats (provided by the experimental animal center of Third Military Medical University), reared in the experimental animal center of Medical University Of Chongqing. 40 rats by weight with the concentration of 0.4ml/100g by intraperitoneal injection of 1% pentobarbital sodium anesthesia, and bilateral oophorectomy, after operation for 3 consecutive days rats were injected with penicillin anti infection. Serum estrogen level in rats detected in January after operation. Serum estrogen level in 11pg/ml screening about 19 rats as successful castrated ovariectomized model.
1.2, 19 ovariectomized rats were randomly divided into experimental group and control group (10 rats in the experimental group and 9 in the control group). Estrogen and placebo were administered respectively. After 4 weeks, the serum estrogen level was detected by chemiluminescence.
1.3 the rats in the experimental group and the control group were tested after 4 weeks of Morris water maze, in order to detect the spatial reference memory ability of the rats.
After the 1.4Morris water maze test, 6 rats were randomly selected from the experimental group, and 5 rats were randomly selected from the control group. Then the rats were anesthetized, perfused and fixed, and the brains were removed.
The 1.5 hemispheres along the coronal line were cut into 1mm thick continuous brain tissue sections. Each rat were randomly selected on one side of the brain. In order to ensure that all parts of the white matter has the same probability of being selected in selected brain sections were randomly placed in the measuring points, measuring click in place from white soft tissue from each rat 5 electron microscope. Cut 1 60NM thick ultrathin sections were magnified 20000 times randomly selected 12 view pictures of each rat were taken 60 photos.
1.6 using the stereo calculation of rats of experimental group and control group total white matter volume method, the myelinated fibers in the white matter the length density, total length, volume density, total volume, average diameter, average diameter of axons, myelinated nerve fiber diameter and axon diameter, myelinated axon diameter and fiber the diameter ratio, and white matter of different diameter with the results of.2 were compared with the statistical parameters of myelinated nerve fibers
2.1 the results of estrogen level: there was no significant difference in serum estradiol level between the 2 groups after operation (P0.05). The serum estradiol levels of 2 groups after estrogen replacement therapy were statistically different (P 0.01).
The results of 2.2Morris water maze test: the spatial reference memory ability of the rats in the experimental group was significantly higher than that in the control group (P 0.05).
2.3 stereology results: total white matter volume of the two groups of rats, myelinated fibers in the white matter of the length density, total length, volume density, total volume, average diameter, average diameter of axons, myelinated nerve fiber diameter and axon diameter difference, myelinated nerve fiber diameter and axon diameter ratio are there was no significant difference (P0.05). The rats in the experimental group the diameter of cerebral white matter is 1.2~1.4 m in the total length of the myelinated nerve fibers than the control group significantly increased by 46.1% (P 0.05). The rats in the experimental group the average diameter of the cerebral white matter of 0~0.4 m in the myelinated nerve fiber diameter and axon diameter significantly increased than the control group 21.6% (P 0.05).
1. materials and methods
1.1 stereological method was used to calculate the volume density of myelin sheath, the total volume of myelin sheath, the inner circumference of myelin sheath, the outer circumference of myelin sheath, the difference of inner and outer circumference of myelin sheath, the ratio of inner and outer circumference of myelin sheath, and the thickness of myelin sheath in experimental group and control group.
1.2 the correlation.2. results between stereological parameters and Morris water maze behavior parameters were analyzed by bivariate correlation analysis statistical method
2.1 experimental groups of rat brain myelinated fibers in the white matter myelin volume than the control group significantly increased by 27% (P 0.05). The experimental group rat cerebral white matter contrast myelin inside and outside the perimeter control group significantly increased by 12.6% (P 0.05).
2.2 the volume density of myelinated nerve fibers in the white matter of the experimental group and the control group were not significantly different from those of the control group. There was no significant difference in the circumference, circumference, circumference and circumference, and myelin thickness between the experimental group and the control group (P0.05).
2.3 rats in the experimental group the average diameter of white matter is 0~0.4 m in the myelinated nerve fiber diameter and axon diameter than the control group significantly increased by 21.6% (P 0.05). The rats in the experimental group the average diameter of white matter 0.4~0.6 m in myelin and Zhou Changzhi contrast to the control group a significant increase of 20.7% (0.05 P), two rats in the average diameter of white matter 0.6~0.8 m myelin sheaths of myelinated fibers in the inner and outer perimeter difference differences trend in experimental group than in the control group increased 14.3% (P=0.058).
2.4 the total medullary volume of myelinated nerve fibers in the white matter was negatively correlated with the first day latency and the 4 day latency in the Morris water maze (P 0.05).
Full text conclusion
1. estrogen replacement therapy has a positive protective effect on the spatial reference memory ability of ovariectomized rats.
2. the total volume of the experimental group and the control group of rats brain white matter and myelinated fibers in the white matter of the length density, total length, volume density, total volume, average diameter, average diameter of axons, myelinated nerve fiber diameter and axon diameter difference, the total volume of the myelinated fibers, and its diameter the ratio of the diameter of axons and myelinated fibers in the white matter myelin within the perimeter, outer perimeter, inside and outside the perimeter between the inner and outer perimeter ratio, the thickness of myelin basic data for the further use of estrogen replacement therapy to delay the aging process of the brain and provide a scientific basis for the treatment of anti inflammatory demyelinating diseases.
3. the total medullary sheath volume of the myelinated nerve fibers in the white matter of the experimental group was found in the experimental group, and the difference in the internal and external circumference of the myelin sheath was significantly higher than that in the control group.
4. compared with the control group, the experimental group rats, the total length of the myelinated fibers in the white matter was significantly increased 1.2~1.4 m in diameter, in the white matter of 0~0.4 m in diameter of myelinated nerve fibers and axon diameter difference significantly increased, a significant increase in 0.4~0.6 m diameter section peripheral myelin length difference at the same time, there are differences in the trend of 0.6~0.8 m diameter of myelin inner and outer perimeter difference. These results for the total volume of rats in the experimental group than in the control of myelin provide the structural basis for group was significantly increased.
5. it was found that the total medullary volume of myelinated nerve fibers in white matter was correlated with the first day incubation period and the fourth day incubation period of the Morris water maze.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R741

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