大鼠胼胝体有髓神经纤维及MBP的老年性改变研究

发布时间：2018-05-27 18:30

本文选题：大鼠 + 胼胝体　；参考：《重庆医科大学》2009年硕士论文

【摘要】： 衰老是不可避免的生理过程,与衰老有关的疾病给家庭和社会带来了许多问题。这其中,大脑的衰老又尤其引人关注。长久以来的观点是老年大脑存在着神经元的大量死亡,然而,随着新的体视学方法的建立和应用,Pakkenberg和Gundersen发现正常老年脑并不存在显著性大脑皮质神经元的死亡,而老年大脑白质体积下降了28%,随后大量的研究也证实了此发现,对大脑白质的研究开始越来越深入和广泛。由于大脑白质不同区域的解剖结构和功能不同,老年大脑白质特定区域改变与老年大脑功能损害的确切关系仍需深入探究。胼胝体作为两大脑半球对应脑皮质之间联络清晰的、解剖和功能都较为清楚的连合纤维束,越来越多的受到研究者的关注。许多采用影像学技术的研究发现,正常老年和异常老年中都存在着胼胝体的萎缩。他们推测这种萎缩可能是纤维束丢失所导致的。上述研究说明了胼胝体神经纤维尤其是有髓神经纤维对相关脑叶神经元之间的神经冲动相互传导的完整性起着至关重要的作用,因此,胼胝体神经纤维的改变很可能是影响大脑认知功能的结构基础之一。但是即往研究都没有对胼胝体内神经纤维进行准确的定量研究,以反映纤维丢失的情况,原因之一在于缺乏准确定量在三维空间内无序排列的神经纤维的手段。Tang等在国际上首次应用了新的体视学方法对大脑白质神经纤维进行定量研究,从而为神经纤维的三维定量提供了有效的研究手段。因此,本研究运用TEM和体视学技术研究Long-Evans大鼠胼胝体及其内有髓神经纤维的老年性改变。众多研究发现,有髓神经纤维受损、丢失的同时,髓鞘蛋白也存在一定变化,并与认知功能降低相关。MBP是CNS特有的一种髓鞘蛋白,对髓鞘的发生和形成具有重要的生理作用。因此,本研究运用Western-blot技术研究大鼠胼胝体MBP的老年性改变,以及MBP的改变与有髓神经纤维改变之间的关系。第一部分 1材料与方法 1.1雌性Long-Evans大鼠,年轻组(7月龄)5只、中老年组(18月龄)5只和老年组(27月龄)6只,体重280±20 g。 1.2每只大鼠随机抽取一侧大脑半球,从中随机抽取6块胼胝体组织块作成超薄切片,从每张切片上随机抽取4个视野在透射电镜下放大6000倍拍照,每只动物共拍摄24张照片。 1.3运用体视学方法分别计算出每只大鼠胼胝体体积、胼胝体有髓神经纤维长度密度和总长度、胼胝体有髓神经纤维体积密度和总体积、髓鞘体积密度和总体积、有髓神经纤维平均直径。 2结果 2.1电镜下,老年组大鼠胼胝体有髓神经纤维存在明显的脱髓鞘的改变,可见双层髓鞘形成、髓鞘板层分离和髓鞘内空泡化。 2.2年轻组大鼠胼胝体体积为70.7±5.6 mm3,中老年组为63.2±4.2 mm3,老年组为51.6±2.6 mm3。与年轻组相比,中老年组显著降低了10.6% (p0.01),老年组显著降低了26.9% (p0.01)。老年组与中老年组相比,胼胝体体积显著降低了18.3% (p0.01)。 2.3年轻组大鼠有髓神经纤维总长度为78.3±7.7 km,中老年组为75.7±11.0 km,老年组为62.4±5.8 km。与年轻组相比,中老年组的下降没有显著性(p0.05),老年组显著下降了20.3% (p0.05)。老年组与中老年组相比,有髓神经纤维总长度显著下降了17.5% (p0.05)。 2.4年轻组大鼠有髓神经纤维总体积为46.6±3.7 mm3,中老年组为43.4±3.0 mm3,老年组为39.4±2.5 mm3。与年轻组相比,中老年组的下降没有显著性(p0.05),老年组显著下降了15.5% (p0.01)。老年组与中老年组相比,下降没有显著性(p0.05)。 2.5年轻组有髓神经纤维髓鞘总体积为21.3±2.5 mm3,中老年组为19.7±2.6 mm3,老年组为18.4±1.4 mm3。三者之间没有显著性改变(p0.05)。 2.6年轻组有髓神经纤维平均直径为0.62±0.03μm,中老年组为0.66±0.53μm,老年组为0.70±0.04μm。与年轻组相比,中老年组有髓神经纤维直径的增加没有显著性(p0.05),老年组显著增加了13.0% (p0.01)。老年组与中老年组相比,没有显著性差异(p0.05)。 2.7与年轻组相比,老年组大鼠直径为0.2 - 0.4μm的有髓神经纤维总长度显著下降了47.3% (p0.01),直径为0.4 - 0.6μm的有髓神经纤维总长度显著下降了35.6% (p0.01)。中老年组与年轻组相比,0.2 - 0.4μm和0.4 - 0.6μm直径段的有髓神经纤维总长度的变化没有显著性(p0.05)。第二部分 1材料与方法 1.1雌性Long-Evans大鼠,7月龄10只,随机分为两组;27月龄10只,随机分为两组。 1.2取7月龄和27月龄大鼠各一组,进行电子显微镜切片制作和体视学分析,方法同第一部分。 1.3取7月龄和27月龄大鼠各一组,从每只动物大脑胼胝体内提取MBP,Western-blot检测MBP表达,实验至少重复5次。 1.4 Quantity One图像分析软件分析蛋白条带OD值,以各蛋白条带与β-actin的灰度值之比表示该蛋白的表达量。 2结果 2.1老年组与年轻组相比,大鼠胼胝体有髓神经纤维总长度下降了20.3% (p 0.01)。有髓神经纤维总体积下降了15.5 (p 0.01)。髓鞘总体积虽有下降,但没有显著性(p0.05)。 2.2与年轻组相比,老年组胼胝体内各分子量的MBP表达量都有降低,但只有Mr 21.5×104的MBP降低具有显著性(p0.05)。对Mr 21.5×104的MBP含量改变与胼胝体有髓神经纤维总长度、总体积和髓鞘总体积作Spearman相关分析,Mr 21.5×104MBP的降低与有髓神经纤维总体积的降低存在正相关(r = 0.770;p0.01),与有髓神经纤维髓鞘的总体积的降低存在正相关(r = 0.636;p0.05),与有髓神经纤维总长度没有明显相关性(r = 503;p0.05)。做多重线性回归分析后发现,随着年龄的增加,MBP的降低与有髓神经纤维髓鞘总体积的降低具有显著的相关性(β= -1.105;p0.05),与有髓神经纤维总长度及有髓神经纤维总体积的降低不具有显著相关性(β= -0.438, p0.05;β= -1.306, p0.05)。全文结论 1.本研究首次把新的体视学方法和电子显微镜技术结合起来定量研究大鼠胼胝体及其有髓神经纤维的老年改变。 2.首次获得了年轻、中老年及老年雌性大鼠胼胝体体积、胼胝体有髓神经纤维的总长度和总体积、髓鞘总体积及纤维平均直径的基本数值,这些数值为今后运用该动物模型探讨CNS退化性疾病对胼胝体有髓神经纤维的影响提供了重要的参考数据。 3.证实了以前在人胼胝体的研究发现,即存在老年性胼胝体萎缩。 4.首次发现老年雌性Long-Evans大鼠大脑胼胝体有髓神经纤维总长度和总体积显著性降低,且总长度的降低主要是由于细小直径的有髓神经纤维降低所致。 5.证实了老年胼胝体MBP的表达降低,并得出胼胝体MBP的表达降低与胼胝体有髓神经纤维的老年改变之间存在相关性。
[Abstract]:Aging is an inevitable physiological process, and aging related diseases have brought many problems to family and society. Among them, brain aging is particularly attractive. The long-standing view is that there is a large number of neuronal deaths in the brain of the elderly. However, with the establishment and application of new stereological methods, Pakkenberg and Gundersen have found that There is no significant cerebral cortical neuron death in the normal aged brain, and the volume of white matter in the aged brain drops by 28%. Subsequently, a large number of studies have also confirmed the discovery that the study of white matter in the brain is becoming more and more deep and extensive. The specific areas of white matter in the brain are changed because of different anatomical structures and functions in different regions of the brain white matter. The exact relationship between the changes and the impairment of brain function in the elderly still needs to be explored. The corpus callosum is more and more concerned with the researchers' attention as a clear, clear, anatomical and functional connection between the two cerebral hemispheres that correspond to the cerebral cortex. Many studies using imaging techniques have found that both normal and abnormal old age are stored. They speculate that the atrophy of the corpus callosum may be caused by loss of fiber bundles. These studies show that the corpus callosum fibers, especially the myelinated nerve fibers, play a vital role in the integrity of the mutual conduction of the nerve impulses between the related lobar neurons. Therefore, the changes in the corpus callosum fibers are likely to be altered. It is one of the structural foundations that affect the cognitive function of the brain. However, there is no accurate quantitative study of the nerve fibers in the corpus callosum to reflect the loss of fibers. One of the reasons is that the lack of accurate quantitative and unordered neural fibers in the three-dimensional space of the hand segment.Tang, for the first time, has applied new stereopsis to the world for the first time. A quantitative study of white matter nerve fibers in the brain provides an effective method for the three-dimensional quantification of nerve fibers. Therefore, this study uses TEM and stereological techniques to study the age-related changes in the corpus callosum and its intramedullary nerve fibers in Long-Evans rats. At the time, the myelin protein is also changed, and associated with the decrease of cognitive function,.MBP is a kind of myelin protein that is unique to CNS. It has an important physiological role in the occurrence and formation of myelin sheath. Therefore, this study uses Western-blot technique to study the age-related changes of the corpus callosum MBP in rats, and the change of MBP with the changes of myelinated nerve fibers. Relationship.
Part one
1 materials and methods
1.1 female Long-Evans rats, young group (7 month old) 5, middle and old age group (18 month old) 5 and old age group (27 month old) 6, weight 280 280 20 G.
1.2 each rat was randomly selected from one side of the cerebral hemisphere. 6 pieces of corpus callosum were randomly selected for ultrathin sections. From each slice, 4 fields were randomly selected to enlarge 6000 times under transmission electron microscope. Each animal was photographed with 24 photographs.
1.3 the volume of corpus callosum, the length density and total length of the myelinated nerve fibers in the corpus callosum, the volume density and total volume of the myelinated nerve fibers in the corpus callosum, the medullary volume density and the total volume, and the average diameter of the myelinated nerve fibers were calculated by the stereological method.
2 Results
2.1 under electron microscope, there were obvious demyelinating changes in the callosal nerve fibers of the aged rats, and the formation of the double myelin sheath, the separation of the myelin sheath and the vacuolation in the myelin sheath.
The volume of corpus callosum in 2.2 young rats was 70.7 + 5.6 mm3, middle and old age group was 63.2 + 4.2 mm3, aged group was 51.6 + 2.6 mm3., compared with young group, the middle and old age group decreased significantly (P0.01), and the elderly group decreased by 26.9% (P0.01). The volume of corpus callosum was significantly reduced by 18.3% (P0.01) in the elderly group compared with the middle and old age group.
The total length of the myelinated nerve fibers in the 2.3 young rats was 78.3 + 7.7 km, the middle and old age group was 75.7 + 11 km, the elderly group was 62.4 + 5.8 km., compared with the young group, the decrease of the elderly group was not significant (P0.05), and the elderly group decreased significantly (P0.05). The total length of the myelinated nerve fibers in the elderly group was significantly decreased by 17.5% (p0.) compared with the middle and old age group. 05).
The total volume of myelinated nerve fibers in 2.4 young rats was 46.6 + 3.7 mm3, middle and old age group was 43.4 + 3 mm3, and aged group was 39.4 + 2.5 mm3., compared with young group, the decrease of middle aged and elderly group was not significant (P0.05), and the elderly group decreased significantly (P0.01). The decline was not significant (P0.05) in the elderly group compared with the middle and old age group.
The total medullary sheath volume of myelinated nerve fibers in the 2.5 young groups was 21.3 + 2.5 mm3, and the middle and old age group was 19.7 + 2.6 mm3, and the elderly group was 18.4 + 1.4 mm3. three. There was no significant change (P0.05).
The average diameter of the myelinated nerve fibers in the 2.6 young group was 0.62 + 0.03 m, the middle and old age group was 0.66 + 0.53 mu m, the elderly group was 0.70 + 0.04 micron M., compared with the young group, there was no significant increase in the diameter of the myelinated nerve fibers in the middle and old age group (P0.05), and the elderly group increased by 13% (P0.01). There was no significant difference in the elderly group compared with the middle and old age group (P0.05).
2.7 compared with the young group, the total length of the myelinated nerve fibers in the aged group of 0.2 - 0.4 mu m was significantly decreased by 47.3% (P0.01). The total length of the myelinated nerve fibers with the diameter of 0.4 - 0.6 m decreased significantly by 35.6% (P0.01). The total length of the myelinated nerve fibers in the middle and old age group was compared with the young group, 0.2 to 0.4, m and 0.4 - 0.6 m diameter segments. The change was not significant (P0.05).
The second part
1 materials and methods
1.1 female Long-Evans rats, 7 month old 10, were randomly divided into two groups. 27 month old 10 rats were randomly divided into two groups.
1.2 each group of 7 month old and 27 month old rats was subjected to electron microscope sectioning and stereology analysis, the same as the first part.
1.3 take 7 month old and 27 month old rats in each group, extract MBP from the corpus callosum of each animal, detect the expression of MBP by Western-blot, and test at least 5 times.
1.4 the Quantity One image analysis software was used to analyze the OD value of protein bands, and the ratio of the protein bands to the gray value of the beta -actin was used to express the protein expression.
2 Results
2.1 the total length of the myelinated nerve fibers in the corpus callosum of the aged group decreased by 20.3% (P 0.01) compared with the young group. The total volume of myelinated nerve fibers decreased by 15.5 (P 0.01). Although the total volume of myelin sheath decreased, the total medullary volume decreased, but was not significant (P0.05).
2.2 compared with the young group, the MBP expression of each molecular weight in the corpus callosum in the elderly group decreased, but only the Mr 21.5 x 104 MBP decreased significantly (P0.05). The MBP content of Mr 21.5 x 104 was associated with the total length of the myelinated nerve fibers in the corpus callosum, the total volume of the myelin sheath and the total medullary volume, and the decrease of Mr 21.5 x 104MBP and myeloid God. There was a positive correlation (r = 0.770; P0.01) with the decrease of total volume of fiber (r = 0.636; P0.05), and there was no significant correlation with the total length of myelinated nerve fibers (r = 503; P0.05). After multiple linear regression analysis, the decrease of MBP and myelinated nerve fibers as age increased. The reduction of total medullary volume has a significant correlation (beta = -1.105; P0.05). There is no significant correlation with the total length of myelinated fibers and the reduction of total medullary nerve fibers (beta = -0.438, P0.05; beta = -1.306, P0.05).
Full text conclusion
1. in this study, the new stereology method and electron microscopy technique were first applied to quantitatively study the aging changes of corpus callosum and myelinated nerve fibers in rats.
2. the corpus callosum volume, the total volume and total volume of the callosal nerve fibers, the total volume of myelin sheath and the average diameter of the fibers were obtained for the first time in young, middle-aged and old female rats. These values provide important implications for the future use of the animal model to explore the effects of CNS degenerative diseases on the callosum myelinated nerve fibers. Reference data.
3. it is confirmed that previous studies in human corpus callosum showed age-related atrophy of corpus callosum.
4. the total length and total volume of the medullary nerve fibers in the corpus callosum of the aged female Long-Evans rats decreased significantly for the first time, and the decrease in the total length was mainly due to the decrease of the myelinated nerve fibers in the small diameter.
5. it was confirmed that the expression of MBP in the corpus callosum of aged patients was reduced, and the correlation between the decrease of MBP expression in corpus callosum and the age-related changes of corpus callosum myelinated nerve fibers was found.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R329.54

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