视神经挫伤后RGCs形态学及其GFAP表达变化规律的研究

发布时间：2018-03-02 04:04

本文关键词： 视神经/损伤视网膜神经节细胞 GFAP 时间相关性　出处：《华中科技大学》2006年硕士论文　论文类型：学位论文

【摘要】： 【研究背景】视神经挫伤是法医学活体鉴定和临床眼科中一种常见的眼外伤,往往给伤者带来不可逆转的视功能损害,正确认识和评价这种损伤对视功能的影响是临床和法医工作者的重要任务。神经胶质原纤维酸性蛋白(glial fibrillary acid protein,GFAP)是构成细胞骨架中间丝的主要成分,主要存在于中枢神经系统的星形胶质细胞中[1]。神经系统损伤时胶质细胞功能的改变常表现为胶质细胞免疫反应性的改变。在许多神经变性疾病均发现GFAP免疫反应性的改变,它被认为是中枢神经损伤的灵敏指示剂。同时,一系列的动物模型研究证实,视神经损伤后视功能下降的病理基础是以视网膜神经节细胞(Retinal ganglion cells, RGCs)的继发性丧失为主[2]。所以,正确认识视神经损伤后病理变化的特点就成了本研究的主要内容。【目的】本实验通过建立大鼠视神经夹挫伤模型,观察伤后RGCs的形态学改变及Müller细胞合成的GFAP的变化,为研究视神经损伤后法医学鉴定提供分子病理学基础。【材料与方法】Wistar成年大鼠45只,分为7组,每组动物5只,参照文献的方法,制成视神经夹挫伤动物模型,伤后各组分别饲养存活1d、3d、5d、7d、9d、14d、21d,同期行灌流固定及眼球取材固定切片。光镜下观察视网膜神经节细胞的改变,免疫组化方法检测不同时间RGCs表达GFAP的水平,对视网膜神经节细胞及GFAP表达变化的规律与损伤时间的关系进行分析。【结果】视神经损伤后诱导视网膜神经节细胞丧失,视神经损伤后1d,可见少量RGCs开始溃变死亡,3d时RGCs开始大量减少,7d时减少约36%,14d时减少约44%,21d时减少约52%。7d之前细胞数目快速减少,14d后呈慢速减少,14d到21d内细胞丢失数目仅为7.43%,而正常组RGCs没有任何明显变化。视神经损伤后1d视网膜GFAP染色与正常大鼠视网膜相似;伤后3d,视网膜神经纤维层和节细胞层有增多的GFAP阳性染色,内网状层、内核层、外网状层可见线条状GFAP阳性着色;伤后7d,节细胞层及内、外界膜之间的GFAP阳性染色进一步加深、密集,并达到高峰;伤后9d,GFAP表达下降,但下降缓慢,已趋于平缓,14d后下降至接近正常水平。【结论】从视网膜病理改变结果看出:与之前研究结果相比,随着致伤强度增加,RGC丢失加重,说明不同的致伤强度造成不同程度的视神经损伤,致伤强度和损伤程度成负相关。实验发现视神经损伤后3d时RGCs开始大量丧失,7d内迅速减少,到14d时已达44%,14d后丧失速度明显放慢,21d后几乎无明显变化,提示在视神经外伤后,RGCs的丧失与时间有明显相关性,这与国外学者Berkelaar等[3]的研究结果相一致。视神经夹挫伤使视网膜GFAP免疫反应性于伤后3d增加,7d达到高峰,表明视神经损伤引起视网膜胶质细胞反应性增高,随时间进展又逐渐下降,于14d后降至正常水平,这与受损RGCs的溃变、死亡率的逐渐增高是一致的。同时实验观察到GFAP的表达部位主要集中在RGCs的胞膜及其纤维的周围,表明GFAP蛋白与RGCs的存活数量密切相关,随着GFAP表达的减弱,受损RGCs不断溃变和死亡,表达也随之减弱。综上所述,视神经损伤后直接机械损伤作用导致视功能迅速下降,继发性的RGCs丧失和轴突的溃变导致视功能的恶化并显示出时间相关性;GFAP蛋白表达水平也表现出一定的时间相关性,其表达强弱与RGCs存活数量多少也密切相关。这些为正确了解视神经损伤后视功能变化的病理因素和预后评估提供了重要的理论基础,也为视神经挫伤的鉴定时机提供了依据。
[Abstract]:[background] optic nerve contusion is a common ocular trauma in forensic science in vivo identification and Clinical Ophthalmology, often to bring injured visual function damage is irreversible, affect the correct understanding and evaluation of the damage of visual function is an important task in clinical and forensic medicine. Glial fibrillary acidic protein (glial fibrillary acid protein. GFAP) is the main component of cytoskeletal intermediate filaments, injury of glial cell function of [1]. neural system mainly exists in the central nervous system in astrocytes glial cell changes are often immune reactive changes. In many neurodegenerative diseases were found GFAP immunoreactivity changes, it is considered to be sensitive the indicator of central nervous system injury. At the same time, a series of studies in animal models confirmed that the pathological basis of optic nerve damage visual function decline in the retina of God The secondary loss of Retinal ganglion cells (RGCs) is mainly [2].. Therefore, correctly recognizing the characteristics of pathological changes after optic nerve injury has become the main content of this research.
[Objective] optic nerve crush model in rats was established by this experiment, the change of RGCs morphology and M ller cell GFAP synthesis was observed after injury of optic nerve injury in forensic medicine provides the molecular pathologic basis.
[materials and methods] 45 adult Wistar rats were divided into 7 groups, each group of animal 5, according to the literature method, made of optic nerve crush injury animal model, each group were reared 1D 3D, 5D, survival, 7d, 9D, 14d, 21d, underwent perfusion fixation and eyeball were fixed slice. Under the light microscope observation on retinal ganglion cell changes, immunohistochemical method to detect the expression level of GFAP RGCs in different time, we analyzed the relationship between law and time of injury on the expression of retinal ganglion cells and GFAP.
[results] after optic nerve injury induced by retinal ganglion cell loss after optic nerve injury, 1D, a small amount of RGCs degeneration death, 3D RGCs began to decrease by about 36% 7d, reduced by about 44% 14d, about 52%.7d before the cell number decreased rapidly decreased 21d, 14d after a slow decrease, 14d 21d cell loss is only 7.43%, and no significant change in normal group. RGCs of 1D after optic nerve injury of retina GFAP staining and normal rat retina; 3D after injury, the retinal nerve fiber layer and ganglion cell layer with positive staining of GFAP increased, the inner plexiform layer, inner nuclear layer, outer plexiform layer visible line GFAP staining; 7d after injury, and the ganglion cell layer, GFAP positive staining between the outside membrane further, dense, and reached the peak at 9D after injury; and the expression of GFAP decreased, but the decline has tended to slow, gentle, after 14d decreased to normal water Ping.
[Conclusion] from the retinal pathological changes showed that compared with the previous research results, with the injury intensity increases, the loss of RGC increased, the intensity of injuries caused by different show different degrees of optic nerve injury, negatively related to the intensity of injuries and damage degree. The experimental results showed that optic nerve injury after 3D RGCs lost a lot, quickly reduce 7d, 14d has reached 44%, after the loss of 14d slowed down significantly, 21d almost no obvious change, suggesting that in the optic nerve after injury, loss of RGCs have significant correlation with time, consistent with the results of the research of foreign scholar Berkelaar [3]. Optic nerve crush GFAP immunoreactive retinal the increase in 3D after injury, reached the peak at 7d, that caused the increase of retinal glial cell reaction of optic nerve injury, over time and gradually decreased, after 14d decreased to normal level, which was associated with impaired RGCs degeneration, mortality by Gradually increase is consistent. At the same time around the observed expression site of GFAP mainly focus on the cell membrane and RGCs fiber, GFAP protein and RGCs showed that the number of survival is closely related with the decreased expression of GFAP, RGCs has damaged the degeneration and death, expression is weaken. To sum up, resulting in direct effect the mechanical damage of visual function after optic nerve injury decreases rapidly, secondary loss of RGCs and axonal degeneration leads to the deterioration of visual function and displays the time correlation; the expression level of GFAP protein also showed a certain time correlation, the expression intensity of RGCs and survival is closely related to the number. These provide an important theoretical basis of pathology the factors and prognosis of optic nerve function changes after its injury in order to correctly understand, also provides the basis for the identification of the timing of optic nerve contusion.

【学位授予单位】：华中科技大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：D919

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