本文關(guān)鍵詞： 中老年卵巢切除大鼠 白質(zhì) 髓鞘 雌激素替代治療 體視學(xué)　出處：《重慶醫(yī)科大學(xué)》2014年碩士論文　論文類型：學(xué)位論文

【摘要】：體視學(xué)研究表明,老年大腦存在白質(zhì)體積的顯著性下降,并且主要是由于白質(zhì)內(nèi)有髓神經(jīng)纖維的總長度下降引起的。并有研究表明，老年大腦存在認(rèn)知功能的障礙，而女性在絕經(jīng)后，雌激素的缺乏更會(huì)加速其認(rèn)知功能下降的進(jìn)程和增加其罹患阿爾茨海默氏�。ˋlzheimer’sdisease，AD）的風(fēng)險(xiǎn)。有髓神經(jīng)纖維和髓鞘異常的結(jié)構(gòu)是認(rèn)知功能下降的重要原因。中樞神經(jīng)系統(tǒng)作為雌激素的靶器官，已有眾多的研究顯示：雌激素可以改善大腦的認(rèn)知能力和空間參考記憶能力。雌激素替代治療能改善絕經(jīng)后婦女包括白質(zhì)在內(nèi)的大腦結(jié)構(gòu)。長期補(bǔ)充雌激素還能防護(hù)老年的白質(zhì)丟失�？梢�，雌激素與大腦衰老和白質(zhì)內(nèi)有髓神經(jīng)纖維及髓鞘之間的關(guān)系甚密。在本實(shí)驗(yàn)中，，我們對(duì)中老年雌性Sprague-Dawley（SD）大鼠行雙側(cè)卵巢切除術(shù)旨在模擬人類絕經(jīng)后雌激素缺乏狀態(tài)下大腦白質(zhì)內(nèi)有髓神經(jīng)纖維及髓鞘結(jié)構(gòu)異常的條件，并運(yùn)用電鏡技術(shù)及體視學(xué)三維精確定量方法研究雌激素替代治療對(duì)卵巢切除大鼠白質(zhì)及白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘超微結(jié)構(gòu)的作用，以了解雌激素對(duì)老年大腦白質(zhì)和脫髓鞘病變的影響。本實(shí)驗(yàn)結(jié)果將為以后進(jìn)一步運(yùn)用雌激素替代治療延緩大腦衰老進(jìn)程和防治脫髓鞘疾病提供科學(xué)依據(jù)。 第一部分1材料與方法 1.1中老年(10~12月齡)雌性SD大鼠40只(第三軍醫(yī)大學(xué)實(shí)驗(yàn)動(dòng)物中心提供),在重慶醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物中心飼養(yǎng)。將40只大鼠按體重以0.4ml/100g的濃度腹腔注射1%的戊巴比妥鈉進(jìn)行術(shù)前麻醉，然后行雙側(cè)卵巢切除術(shù)，術(shù)后連續(xù)3天對(duì)大鼠注射青霉素以抗感染。術(shù)后1月檢測(cè)大鼠的血清雌激素水平。篩選出血清雌激素水平在11pg/ml左右的19只大鼠作為成功去勢(shì)的卵巢切除模型。 1.2將篩選出的19只卵巢切除大鼠隨機(jī)分為實(shí)驗(yàn)組與對(duì)照組（實(shí)驗(yàn)組10只，對(duì)照組9只），分別給予雌激素與安慰劑處理，4周后再次運(yùn)用化學(xué)發(fā)光法檢測(cè)血清雌激素水平。 1.3實(shí)驗(yàn)組與對(duì)照組大鼠在4周后進(jìn)行Morris水迷宮行為學(xué)測(cè)試，以檢測(cè)大鼠的空間參考記憶能力。 1.4Morris水迷宮測(cè)試結(jié)束后，從實(shí)驗(yàn)組隨機(jī)抽取6只大鼠，從對(duì)照組隨機(jī)抽取5只大鼠。然后對(duì)大鼠進(jìn)行麻醉、灌注、固定，取出大腦。 1.5將左右大腦半球沿冠狀線分別切成1mm厚的連續(xù)腦組織切片。每只大鼠隨機(jī)抽取一側(cè)大腦半球。為保證白質(zhì)的各個(gè)部位都有相同的概率被抽取，在抽取的腦組織切片上隨機(jī)疊放測(cè)點(diǎn)，在測(cè)點(diǎn)擊中的地方抽取白質(zhì)組織塊，每只大鼠抽取5塊電鏡標(biāo)本。切取1張60nm厚的超薄切片，放大20000倍后隨機(jī)選取12個(gè)視野進(jìn)行拍照，每只大鼠共拍得60張照片。 1.6運(yùn)用體視學(xué)方法計(jì)算實(shí)驗(yàn)組和對(duì)照組大鼠白質(zhì)總體積，白質(zhì)內(nèi)有髓神經(jīng)纖維長度密度、總長度、體積密度、總體積、平均直徑、軸突的平均直徑、有髓神經(jīng)纖維直徑及其軸突直徑之差、有髓神經(jīng)纖維直徑及其軸突直徑之比，并對(duì)不同直徑范圍的白質(zhì)內(nèi)有髓神經(jīng)纖維各統(tǒng)計(jì)參數(shù)進(jìn)行比較。2結(jié)果 2.1雌激素水平的結(jié)果：術(shù)后檢測(cè)的2組大鼠血清雌激素水平差異無統(tǒng)計(jì)學(xué)意義（P0.05）。雌激素替代治療后檢測(cè)的2組大鼠血清雌激素水平差異具有統(tǒng)計(jì)學(xué)意義（P 0.01）。 2.2Morris水迷宮測(cè)試結(jié)果：實(shí)驗(yàn)組大鼠的空間參考記憶能力較對(duì)照組顯著性提高（P 0.05）。 2.3體視學(xué)結(jié)果：兩組大鼠的白質(zhì)總體積、白質(zhì)內(nèi)有髓神經(jīng)纖維長度密度、總長度、體積密度、總體積、平均直徑、軸突的平均直徑、有髓神經(jīng)纖維直徑及其軸突直徑之差、有髓神經(jīng)纖維直徑及其軸突直徑之比均沒有顯著性差異（P0.05）。實(shí)驗(yàn)組大鼠在直徑為1.2~1.4μm的大腦白質(zhì)內(nèi)有髓神經(jīng)纖維總長度比對(duì)照組顯著性增加了46.1%（P 0.05）。實(shí)驗(yàn)組大鼠在平均直徑為0~0.4μm的大腦白質(zhì)內(nèi)有髓神經(jīng)纖維直徑及其軸突直徑之差比對(duì)照組顯著性增加了21.6%（P 0.05）。 1.材料與方法 1.1運(yùn)用體視學(xué)方法計(jì)算實(shí)驗(yàn)組與對(duì)照組大鼠白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘的體積密度、髓鞘的總體積、髓鞘的內(nèi)周長、髓鞘的外周長、髓鞘的內(nèi)外周長之差、髓鞘的內(nèi)外周長之比、髓鞘的厚度。 1.2運(yùn)用雙變量相關(guān)分析統(tǒng)計(jì)學(xué)方法分析體視學(xué)參數(shù)和Morris水迷宮行為學(xué)參數(shù)之間的相關(guān)性。2.結(jié)果 2.1實(shí)驗(yàn)組大鼠大腦白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘總體積比對(duì)照組顯著性增加了27%(P 0.05)。實(shí)驗(yàn)組大鼠大腦白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘內(nèi)外周長之差比對(duì)照組顯著性增加了12.6%（P 0.05）。 2.2實(shí)驗(yàn)組與對(duì)照組大鼠大腦白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘的體積密度、內(nèi)周長、外周長、內(nèi)外周長之比、髓鞘的厚度均無統(tǒng)計(jì)學(xué)差異（P0.05）。 2.3實(shí)驗(yàn)組大鼠在平均直徑為0~0.4μm的白質(zhì)內(nèi)有髓神經(jīng)纖維直徑及其軸突直徑之差比對(duì)照組顯著性增加了21.6%（P 0.05）。實(shí)驗(yàn)組大鼠在平均直徑為0.4~0.6μm的白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘內(nèi)外周長之差比對(duì)照組顯著性增加了20.7%（P 0.05），兩組大鼠在平均直徑為0.6~0.8μm的白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘內(nèi)外周長之差存在差異性趨勢(shì)，實(shí)驗(yàn)組較對(duì)照組增加了14.3%（P=0.058）。 2.4白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘總體積與Morris水迷宮第1天潛伏期及第4天潛伏期均呈負(fù)相關(guān)（P 0.05）。 全文結(jié)論 1.雌激素替代治療對(duì)中老年卵巢切除大鼠的空間參考記憶能力具有積極的保護(hù)作用。 2.獲得了實(shí)驗(yàn)組和對(duì)照組大鼠大腦白質(zhì)總體積，白質(zhì)內(nèi)有髓神經(jīng)纖維長度密度、總長度、體積密度、總體積、平均直徑、軸突的平均直徑、有髓神經(jīng)纖維直徑及其軸突直徑之差、有髓神經(jīng)纖維直徑及其軸突直徑之比以及白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘的總體積、內(nèi)周長、外周長、內(nèi)外周長之差、內(nèi)外周長之比、髓鞘的厚度等基本數(shù)據(jù)，為以后進(jìn)一步運(yùn)用雌激素替代治療延緩大腦衰老進(jìn)程和防治脫髓鞘疾病提供了科學(xué)依據(jù)。 3.發(fā)現(xiàn)實(shí)驗(yàn)組大鼠大腦白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘總體積、髓鞘的內(nèi)外周長之差較對(duì)照組顯著性增加。 4.發(fā)現(xiàn)與對(duì)照組相比，實(shí)驗(yàn)組大鼠在1.2~1.4μm直徑段的白質(zhì)內(nèi)有髓神經(jīng)纖維總長度顯著性增加，在0~0.4μm直徑段的白質(zhì)內(nèi)有髓神經(jīng)纖維直徑及其軸突之差顯著性增加，在0.4~0.6μm直徑段的髓鞘內(nèi)外周長之差顯著性增加，同時(shí)在0.6~0.8μm直徑段的髓鞘內(nèi)外周長之差存在差異性趨勢(shì)。這些研究結(jié)果為實(shí)驗(yàn)組大鼠髓鞘總體積較對(duì)照組顯著性增加提供了結(jié)構(gòu)基礎(chǔ)。 5.發(fā)現(xiàn)白質(zhì)內(nèi)有髓神經(jīng)纖維髓鞘總體積與Morris水迷宮第1天潛伏期和第4天潛伏期具有相關(guān)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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741

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