【作者】 杨明；

【导师】 陈正形；

【作者基本信息】 浙江大学 ， 外科学， 2004， 博士

【摘要】 第一部分：马尾神经受压大鼠马尾与脊髓圆锥神经细胞的形态结构变化 目的：通过对大鼠马尾受压后其形态结构的变化及脊髓圆锥神经元的形态结构变化的观察，证实马尾受压可导致脊髓圆锥神经元的损害，寻找马尾综合征的可能病因机制。 方法：SD大鼠66只，雄性，300—350克，分术后30min、2h、4h、8h、1d、3d、1w、2w、3w取材，及假手术组与正常对照组共11组。 动物模型的制作：参照Kawakami等的方法，用质量浓度为0.4％的硫贲妥钠（7mg／kg）腹腔注射麻醉，剪去术区被毛，75％酒精消毒，无菌手术暴露L₅节段棘突与椎板，蚊式血管钳咬除L₅棘突与椎板，暴露硬膜，于硬膜椎板间隙向椎管头端塞入一条1×2×10mm³的硅酮片。待动物苏醒后，均为出现截瘫，按改良Tarlov法（Salzman评分标准）测定神经功能。浙江大学博士毕业生论文飞 取材动物在各时相点先行改良Tarlov法神经功能评分后麻醉，经 左心室置主动脉插管，剪破右心耳，以0.9%生理盐水灌注，待右心耳流出 透明液体后，改用4%的多聚甲醛灌流15一20分钟，待大鼠尸体僵硬后， 完整取下T7以下的脊髓与马尾。切取52一53节段脊髓与受压节段马尾（L.）。 HE染色切片标本制作:取出的脊髓于马尾组织做石蜡切片，片厚 IOum，每块组识随机取连续切片多张，取一套行HE染色。 电镜标本制作:选造模术后3天、1周、2用、3周及假手术组大鼠各 一只，按上述取材法取出脊髓与马尾，将取出的脊髓在解剖显微镜下矢状 切成两半，取马尾的其中一束神经纤维切成Zlnln长度。2.5%戊二醛15ml 进行预固定，2小时后用缓冲液充分漂洗，再用1%饿酸溶液作后固定。固 定过程温度保持在4℃。用乙醇系列脱水。用环氧树脂618包埋液逐步取飞代脱水剂。超薄切片100A。用醋酸铀和构株酸铅进行染色。 结果:压迫组术后即出现双后肢全瘫或不全瘫，，改良Tarlov评分均 为O一2级，3w后除个别恢复为3级外，后肢运动功能无恢复。HE染色结果: 造模术后30min一ld，马尾后方神经束膜与神经束间小血管关闭或狭窄变 形，前方正常。造模术后3d，小血管重新开放，神经纤维髓鞘退变，神经纤 维间水肿，可见炎性细胞浸润。术后lw马尾后方直接受压处神经纤维变性， 结构不清，纤维组织内有肉芽组织形成，马尾腹侧神经纤维变性，髓鞘变 粗，神经内小血管扩张、毛细血管增生，受压脊膜增生。Zw一3w时，上述 变化更为严重。马尾受压后sh时，个别骸髓神经细胞过染，细胞核结构仍 正常;ld后，骸髓前角细胞中的较多细胞出现结构变异，着色异常，细 胞核浓聚，细胞皱缩。电镜观察结果:造模术后3d:髓鞘染色不均匀，边 缘粗糙，髓鞘内裂隙增多。雪旺氏细胞胞核可见异染色质边聚，胞质内小 泡与溶酶体增多。造模术后1一3w上述变化进一步加重。压迫组脊髓前角 神经元表现:造模术后3d起，神经细胞胞核仍为圆形或椭圆形，但明显深 染，核周出现异染色质，线粒体肿胀，少数线粒体见外膜溶解破裂。胞内 出现较多的胞质小泡。术后1，:胞核明显深染，异染色质增多边聚，出现 胞核扭曲固缩，线粒体肿胀明显，峭稀少，有的断裂。粗面内质网断裂、浙江大学博士毕业生论文脱颗粒，胞质内见大量小泡。周围髓鞘松散解体。术后2w、3w组表现与术后1w组相似。 结论:1.马尾受压后可导致脊髓圆锥神经元的形态与结构的改变。2.神经元的损伤可能是造成临床上马尾神经综合症减压术后，神经功能恢复差的原因之一。第二部分马尾神经受压对脊髓圆锥神经元凋亡的影响 目的:采用原位末端脱氧核糖酶转移酶介导dUTP标记（TUNEL）技术，对马尾神经受压的大鼠脊髓圆锥进行检测，以明确神经细胞在马尾神经受压后凋亡速度是否变化以及凋亡细胞数量与时相的关系。 方法:TUNEL染色操作步骤按说明书进行，结果判定:细胞核中有棕黄色颗粒者为阳性细胞，即凋亡的细胞。测脊髓横截面面积。统计学处理:每张切片的阳性细胞数除以脊髓横切面的面积即为单位面积的阳性细胞数，所得数据经 SPSSn.5软件处理，以X士S表示，统计学方法采用单因素方差分析。 结果:TUNEL染色阳性细胞计数结果为:阴性对照:0.28士0.34，假手术组:0.53士0.52，术后3Omin:0.60士0.47，术后Zh:0.83士0.54，术后4h:1.17土0.55，术后sh:4.05士0.95，术后ld:9.22士4.65，术后3d:10.30士2.65，术后lw:6.23士1.96，术后Zw:5.64士1.71，术后3w:5.09士1.58。与对照组相比，术后8h起阳性细胞增加，术后3d为高峰，至术后3w，仍显著高于对照组。 结论:马尾神经受压可导致脊髓圆锥神经元调亡数量的明显增加。受压后8h增加，3d达高峰，3w时仍擒于正常。晚期凋亡不能逆转，这可能是CES逾期手术效果不理想的重要原因之一。本实验的结果提示马尾受压后8h前减压应有良好疗效，8h后解压的疗效需由后续的实验证实。浙江大学博士毕业生论文、) 第三部分大鼠马尾神经受压后脊髓圆锥神经元热休克蛋白70表达 的变化更多还原

【Abstract】 Alteration of structure of cauda equina andneuron in medullary cone in compression injury ofcauda equina in ratsObjective: To document that neuron in medullary cone injuryed when cauda. equinawas compressed through Observationthe structure of cauda equina and neuron in medullary cone in compression injury of cauda equina in rats.Methods: 66 male S-D rats were selected at random, they were classified as 11 groups:normal control ,sham operation, 30min,2h,4h,8h5ld,3d,lw,2w,3w after operation.The cauda equine compression model were created as the method of Kawakami and estimated neurological function as the modified Tarlov’s methold when animal awaked. Animals were anesthesiaed at time above-mentioned, set an aortic cannlation through left ventricle, cuted theright auricle made blood diffluence. 0.9% Saline was instillated until outflow was transparent,changed the instillation fluid to 4% formaldehydum polymerisatum for 15-20min.cuted S2-S3 segment of spinal cord and compressed cauda equine. Those tissue were used for paraffin section and HE stain.Transmission electron microscope sample preparation:each one in five groups:sham operation^ d,lw,2w,and 3w after operation, were selected. Cuted tissue taked out to small pieces, 2.5% glutaral 15ml was used to pre-fix, two hours later, the sample was rinsed by buffer; then post-fixed by 1% osmium acid, enviromental temperature kept 4C;desiccated by ethanol; embed by epoxy resin 618.Ultrathin section was made; at last, the sample was stained by acetic acid U and lead citrate.Results:The animals in groups cauda equinea compressed wre paraplegia. The Scores of modified Tarlov’s mothold were 0-2 grade only, few cases recover to 3 grade. HE stain showed that blood vessles were closed or constricted after compression,in group 3d post-operation,small vessels opened again. Myelin sheath of nerve fibers degenerated, and there was edema, inflammatory cells among nerve fibers. In group lw post-operation, fibrosis was seen in many spots of slids, and small vessles dilataltion, blood capillary proliferation were seen in most cases.To 2-3w post-operation, situation above-mentioned was worser. Individual neurons in spinal cord were dark stained,in those Id post -operation,cells shrunken can been seen.The results of electron microscope observation: Myelin sheath of nerve fibers degeneration can be finded 3d after opretion- Myelin sheath became rough and delamination in post-operation, and it became more serious in following time.There were some changes in neurons in spinal cord anterior horn in 3d after operation, Nuclears were dark stained, Heterochromatin were seen under nuclear membrane, Mitochondrion were swellingln samples lw post-operation, nuclears were pycnosis, mitochondrion were more swelling andthe crest disappeared.The manifestation was same in 2-3w after operation.Conclusions: l.The structure of neurons in medullary cone will alter due to compression injury of cauda equine. 2. The injury of medullary cone neurons maybe one of reasons of poor recovery after cauda equina compressed .SECTION 2:The effect of compressed cauda equine to neuron apoptosis in medullarycone in ratsObjective: To determine if apoptosis cells in medullary cone increase in cauda equina compressed rats and the change pattern with time through TUNEL staining.Methods: The methods of animal model and paraffin section prepareing were mentioned in section 1. TUNEL staining was carried out as the introduction of in situ cell apoptosis detection kit. The outcome’s judgement: positive findings meant that nuclei was stained by brown; if not stained, negative.The area of spinal cord were determined by Photoshop 7.0 program under microscope, amplifying 20x. Statistic analysis:The positive cells in lmm2 were calculated. The data were disposaled by SPSS 11.5 program .One way analysis of variance was used.Results: The number of positive stain cells in lmm2 area in each group:negative control: 0.28+0.34, sham operation: 0.53+0.52, 30min post-operation: 0.60+0.47, 2h更多还原