RCS大鼠视网膜变性过程中α亚型神经节细胞形态学变化的研究

发布时间：2018-06-15 05:22

本文选题：视网膜 + RCS大鼠　；参考：《第三军医大学》2010年硕士论文

【摘要】： 视网膜色素变性(retinitis pigmentosa, RP)是一种由于感光细胞进行性缺失,导致视网膜内各层神经元发生继发变性的严重致盲性疾病,近年来人们已对该病的治疗进行了各种尝试,包括视网膜移植、基因治疗、视觉假体、药物干预等,取得了一定的进展,但由于视网膜色素变性的发病机制尚不明确,因此为了明确其发病机制,寻找有效的治疗手段,我们需要对该疾病进行深入的研究。视网膜神经节细胞(retinal ganglion cells, RGCs)是视网膜内的第三级神经元,也是视网膜发育中第一个产生的神经元,其树突主要接受视网膜内的第二级神经元视锥双极细胞(retinal bipolar cells, RBCs)的信息传递,同时也可通过无长突细胞横向联系;其轴突延伸形成视神经,是视觉传输系统的重要组成部分。RGCs的树突是接受神经信号传入的主要部位,树突的形态发生对突触信号的输入、处理以及神经环路的形成起重要作用。在对慢性高眼压、青光眼、年龄相关性黄斑变性和视网膜脱离等疾病的研究发现,RGCs数量在病变过程中逐渐减少,残留RGCs的树突分支随着病变的发展呈现递减的趋势。对多种视网膜变性动物模型的研究发现,随着感光细胞的死亡,RGCs虽然在数量上出现明显减少,但是存活的RGCs形态并未发生改变并且仍然能诱发出动作电位。既往的研究对视网膜变性过程中RGCs形态是否发生改变意见不一。本实验室既往的研究发现,RCS大鼠(Royal college of surgeon rat, RCS)视网膜变性过程中,RGCs数量在早期有明显减少,其中以胞体横径最大的α亚型减少最为显著,但是这些存活的α亚型RGCs在视网膜变性过程中形态是否发生改变未作进一步研究。综上所述,我们提出如下假设:RCS大鼠视网膜变性过程中,随着感光细胞的不断变性死亡,视锥双极细胞因失去信号输入,其位于内丛状层的与RGCs进行突触联系的轴突分支发生了形态学改变,导致RGCs中对损伤最为敏感的α亚型RGCs在突触形态上形态发生改变。针对以上假设,本文的主要研究内容及研究结果如下: 1、通过对视网膜DiI染色方法进行改进,采用视网膜铺片DiI示踪标记RGCs的方法,从单细胞水平对α亚型神经节细胞树突一二级分支数目、一级分支直径及树突分支频率的变化进行研究,了解RCS大鼠视网膜变性过程中α亚型RGCs树突的形态学变化。结果发现:1)RCS大鼠视网膜变性过程中,P21dα亚型RGCs一级分支直径出现显著增粗,但是在病程的中晚期未见改变。α亚型RGCs一级分支直径为何在RCS大鼠变性早期出现显著增粗,这种改变有何意义尚不清楚,有待于进一步研究。2)RCS大鼠视网膜变性过程中,α亚型RGCs树突分支频率在早期P21、30d有显著增加,而在晚期P60、90d时RGCs的树突分支频率显著减少。树突分支频率是树突发生分叉的次数,反映了树突数量的变化,因此,我们推测在RCS大鼠视网膜变性早期,随着上级神经元数量的减少,为了接受上级神经元的信息传递并与周围神经元形成突触联系,α亚型RGCs代偿性更早地伸出更多的树突,与周围的神经元形成突触联系以维持其功能,在视网膜变性的中晚期,即P60、90d,随着视网膜各级神经元的进行性减少,RGCs上级神元大量缺失使神经信息传入减少,视网膜内残留的α亚型RGCs树突分支也出现大量萎缩丢失,分支数目显著减少。我们的研究既对α亚型RGCs的树突一二级粗大分支数目进行了测量,也对树突的终末分支频率进行了观察,结果表明,在视网膜变性过程中α亚型RGCs树突粗大分支数目没有明显改变,而其终末的细小分支则出现了明显的减少。提示了视网膜变性过程中RGCs树突形态变化主要在终末分支,这种形态学的改变可能与上级神经元视锥双极细胞的轴突数目减少导致的传入信息减少有关。 2、应用免疫组织化学方法,采用Recoverin特异标记2型及8型视锥双极细胞,观察RCS大鼠视网膜变性过程中视锥双极细胞轴突密度的变化。结果发现:RCS大鼠视网膜变性过程中,随着视网膜外核层的感光细胞进行性缺失,Recoverin阳性视锥双极细胞的轴突密度在P30d开始出现减少,P90d减少更为显著。结果提示:失去上一级信号传入后,视锥双极细胞的数目和轴突分支出现减少,这种改变可能对RGCs的信息传入产生影响。综上所述,本课题得出如下结论: 1、采用视网膜铺片DiI荧光染色,可以较好的观察神经节细胞形态,对了解和分析正常及病变中神经节细胞形态特点及变化规律提供了一种简单易行的方法。 2、RCS大鼠视网膜变性过程中,α亚型RGCs树突一、二级分支数目无显著变化,而树突分支频率在变性过程中呈现先增高后降低的趋势。结果提示:RCS大鼠视网膜变性过程中,α亚型RGCs树突形态的改变可能主要是其树突终末分支的萎缩和丢失,而这种形态上的改变可能是由于传入信息减少所致,但是其在功能上发生了怎样的改变有待于进一步研究。 3、RCS大鼠视网膜变性过程中,2型和8型视锥双极细胞轴突密度呈现逐渐递减的趋势。结果提示:在视网膜变性过程中,2型和8型视锥双极细胞可能出现了细胞数量的减少,并且其轴突末梢分支也出现了萎缩和丢失,这种轴突形态上的改变可能对下级神经元RGCs的信息传入产生影响。
[Abstract]:Retinitis pigmentosa (RP) is a serious blinding disease causing secondary degeneration of the neurons in the retina due to the progressive loss of photoreceptor cells. In recent years, many attempts have been made for the treatment of the disease, including retinal transplantation, gene therapy, visual prosthesis, drug intervention and so on. Some progress, but the pathogenesis of retinitis pigmentosa is not clear, so in order to identify its pathogenesis and find effective treatment, we need to study the disease in depth.
The retinal ganglion cell (retinal ganglion cells (RGCs)) is the third stage neuron in the retina, the first neuron produced in the development of the retina, and its dendrite is mainly transmitted by the information transmission of the retinal bipolar cells (RBCs) in the retina of the retina, as well as through the transversal of the amacrine cells. The axon extends to the optic nerve. It is an important part of the visual transmission system, the dendrite of.RGCs is the main part of the nerve signal. The morphogenesis of the dendrite plays an important role in the input of the synaptic signal, the processing and the formation of the nerve loop.
In the study of chronic ocular hypertension, glaucoma, age-related macular degeneration and retinal detachment, the number of RGCs decreased gradually during the lesion, and the dendritic branching of the residual RGCs showed a decreasing trend with the development of the lesion. However, there is a significant decrease in number, but the surviving RGCs morphology does not change and still can induce action potential. Previous studies have disagreed on whether RGCs morphologic changes occurred during retinal degeneration. Previous studies in this laboratory found that RCS rats (Royal College of surgeon rat, RCS) were in the process of retinal degeneration. The number of RGCs decreased significantly in the early stage, among which the largest alpha subtype of the cytosolic transverse diameter decreased most significantly, but the morphologic changes of the surviving alpha subtype RGCs were not further studied in the retinal degeneration process. In summary, we put forward the following hypothesis: in RCS rat retinal degeneration process, with the continuous photoreceptor cells In denatured death, the axonal branch of the conical bipolar cell, which is located in the inner plexiform layer, has a synaptic connection with RGCs, resulting in the loss of signal input, resulting in a change in the morphology of the most sensitive RGCs subtype RGCs in the synapse.
In view of the above assumptions, the main research contents and results are as follows:
1, by improving the retinal DiI staining method and using the method of DiI tracer labeling RGCs, the changes of the number of branches of the dendritic one or two branches, the diameter of the primary branch and the frequency of the dendritic branching were studied from the single cell level to understand the morphology of the RGCs dendrites of the RCS subtype of the retina during the degeneration of the retina of the rat. The results are as follows: 1) in the process of retinal degeneration in RCS rats, the diameter of the primary branch of the P21d alpha subtype RGCs is markedly thickened, but it has not changed in the middle and late stages of the disease. Why is the primary branch diameter of the alpha subtype RGCs in the early stage of the denaturation of the RCS rat obviously thickening, what is the significance of this change is still unclear, and it needs to further study.2) RCS In the process of retinal degeneration, the frequency of the RGCs dendrite branch of the alpha subtype increased significantly in the early P21,30d, and the frequency of the dendritic branching decreased significantly at the late P60,90d. The frequency of the dendritic branching was the number of dendrites, reflecting the changes in the number of dendrites. Therefore, we speculate that in the early stage of retinal degeneration in RCS rats, the frequency of dendrites in the RCS rat was with the superior. The reduction in the number of neurons, in order to accept the information transmission of superior neurons and to form synaptic connections with the surrounding neurons, the alpha subtype RGCs compensatory to extend more dendrites earlier and form synaptic connections with the surrounding neurons in order to maintain its function. In the middle and late stages of retinal degeneration, P60,90d, with the progression of retinal neurons in the retina. The loss of RGCs superior deity resulted in the reduction of neural information, and a large number of atrophy and loss of the branches of the RGCs dendrites in the retina, and the number of branches decreased significantly. Our study not only measured the number of one or two grade coarse branches of the dendrites of the RGCs subtype, but also observed the terminal branch frequency of the dendrites. The results showed that the number of the coarse branches of the RGCs dendrite of the alpha subtype did not change obviously during the degeneration of retina, and the fine branches of the end were obviously reduced. It was suggested that the morphological changes of RGCs dendrites in the process of retinal degeneration were mainly in the terminal branch, which may be related to the axis of the pyramid bipolar cells of the superior neurons. The decrease of the number of inrush is associated with a decrease in the number of sudden inrush.
2, the changes in the axon density of cone bipolar cells in the retinal degeneration process of RCS rats were observed by Recoverin specific labeling of type 2 and type 8 conical bipolar cells. The results showed that in the degeneration process of RCS rats, the Recoverin positive cone bipolar disorder with the absence of the photoreceptor cells in the outer retinal nucleus. The cell axon density decreased at the beginning of P30d, and the P90d decreased more significantly. The results suggest that the number of cone bipolar cells and the axon branches decrease after the loss of the upper level signal, and this change may affect the information afferent of RGCs.
To sum up, the following conclusions are drawn.
1, the morphology of ganglion cells can be well observed by DiI fluorescent staining, which provides a simple and easy way to understand and analyze the morphological characteristics and changes of ganglion cells in normal and pathological changes.
2, in the process of retinal degeneration in RCS rats, the number of RGCs dendrites of alpha subtype has no significant change, but the branch frequency of the dendrite increases first and then decreases in the denaturation process. The results suggest that the modification of the RGCs dendritic morphology of the RCS subtype may be mainly the atrophy and loss of the end branch of the dendrite of the rat retina during the degeneration of retina. This change may be due to the reduction of incoming information, but its functional changes need further study.
3, during the retinal degeneration of RCS rats, the axon density of type 2 and 8 types of cone bipolar cells gradually diminishing. The results suggest that in the process of retinal degeneration, the number of cells in type 2 and 8 types of cone bipolar cells may decrease, and the axon branches are also atrophied and lost, and the morphological changes of this axon are changed. It may affect the incoming information of lower neurons RGCs.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R774.1

【参考文献】