【作者】 张海涛；

【导师】 王一；

【作者基本信息】 第三军医大学 ， 眼科学， 2010， 硕士

【摘要】 视网膜色素变性(Retinitis pigmentosa, RP)是一组以进行性光感受器或色素上皮功能丧失为共同表现的不可逆神经性致盲眼病,是遗传性视觉损害和盲目的最常见原因之一。近年来对于RP在临床及基础研究方面取得一定进展,但对于在视网膜变性过程中各级神经元的病理改变尚不清楚。视网膜水平细胞与视杆双极细胞是视网膜中重要的二级神经元,二者与光感受器在外网状层形成三联体突触和常态突触,在视觉信息的处理与传递中具有关键作用。当视网膜变性发生时,随着光感受器的逐渐凋亡,作为下游神经元的水平细胞与视杆双极细胞首先失去了上游信息的输入,其形态结构会如何变化,其形态的改变是否对下游的第二级突触有无影响。既往有学者对P23H大鼠、视网膜脱离小鼠、rd、rd1、rd10、FVB/N、crx-/-小鼠模型上的研究中发现在变性中期水平细胞树突、视杆双极细胞顶端树突出现萎缩,变性晚期视杆双极细胞与水平细胞产生新的突起并形成了异位突触,异位突触周围有突触标记物环绕。由于既往研究所用动物模型的发病机理各异,得到的结果相差较大,对临床治疗指导作用有限。RCS大鼠是研究视网膜色素变性的经典的动物模型,由于其与人类RP在病因及病程进展等方面有许多相似之处,因此以RCS大鼠为模型来研究视网膜水平细胞与视杆双极细胞在视网膜变性过程中形态学的变化将对RP的临床治疗提供理论基础。近年来视网膜移植、视觉假体和视网膜修复性植入策略是拯救视网膜的两种主要策略,我们认为以上策略能否成功的关键第一是受体视网膜中的二、三级神经元在移植时首先须保持稳定的形态结构,第二是供体组织或细胞能与受体视网膜中的二级神经元产生功能性突触连接,第三选择合适的治疗时间窗。所以本实验结果将对RP的基因和分子治疗时间窗的选择、对植入细胞和假体的稳定性、对治疗性前体细胞的整合等都会提供实验依据、理论基础和新的思路。本研究采用DiI示踪标记结合视网膜冰冻切片和荧光免疫组织化学染色的方法,来研究RCS大鼠视网膜水平细胞及相关神经元在变性过程中形态学的变化。本研究包括两个部分:第一部分:RCS大鼠视网膜水平细胞在变性过程中的形态学变化RCS大鼠在视网膜变性的过程中,光感受器和带状突触的数量都在不断减少,这个过程可以持续到出生后90天,光感受器和带状突触的丢失速率比较均匀。P2MRCS大鼠视网膜外核层厚度仅约为对照组的15%,P3M RCS大鼠视网膜外核层仅残余一层细胞核。P1M、P2M RCS大鼠视网膜水平细胞的轴突终末在外网状层的分层未见明显改变,P3M时出现分层改变,不再呈水平线状,出现新生突起并向外核层延伸。DiI示踪标记水平细胞结果分析表明:P1M、P45d RCS大鼠视网膜水平细胞的树突与轴突终末的形态无明显的改变。P2M RCS大鼠视网膜水平细胞的树突与轴突终末的形态出现明显的改变:树突、轴突野明显变小(P<0.05),树突、轴突终末细小分支丢失,树突、轴突终末分支长度比值(RCS大鼠与正常组大鼠水平细胞的树突、轴突终末分支长度比值)明显变小(P<0.05)。第二部分:RCS大鼠视网膜视杆双极细胞与无长突细胞在视网膜变性过程中形态学的变化。P1M、P45d RCS大鼠视杆双极细胞形态正常,P2M RCS大鼠视杆双极细胞顶端树突萎缩;P3MRCS大鼠视杆双极细胞顶端萌生新的神经突起。无长突细胞的树突在内网状层的分层至变性晚期亦未见改变。本研究结论:1、RCS大鼠视网膜水平细胞与视杆双极细胞在变性过程中形态的改变是由于光感受器的凋亡引起的继发性反应,其形态的稳定依赖于光感受器。2、RCS大鼠视网膜水平细胞与视杆双极细胞形态的改变未影响无长突细胞的树突在内网状层的分层。更多还原

【Abstract】 Retinitis Pigmentosa (RP) is a group of hereditary retinal degeneration characterized by progressive dysfunction of photoreceptors cells (PRC) and associated with progressive cells loss and eventually atrophy of several retinal layers. It is the common reason of hereditary visual impairment and blindness in clinic. So far, we have made progress on the clinical and basic research in recent years, But the various pathological changes of retinal neurons in the course of RP are not clear.Horizontal cell and bipolar cell are the second-order neurons in retinal, they form triad synapses and normal synapses with photoreceptor. They play a key role in visual information processing and transmission. With the gradual reduction of photoreceptors, As the downstream neurons, they begin to lose the upper information input, how they influenced the normal visual information processing and transmission?We would like to know whether the morphology of rod bipolar cell and horizontal cell change when the retinal degeneration occurs. scholars have studied many models on retinal degeneration,such as P23H rat, retinal detachmented mice, rd, rd1, rd10, FVB/N, crx-/- mice, they found that dendrites of rod bipolar cells and horizontal cells shrinked in the medium-term degeneration, they sprouted new processes and formed ectopic synapses late in the degeneration, there were synaptic markers surround around the ectopic synapses. Because there are big difference on pathogenesis betwen previous animal models and human RP, so the guidance of treatment to restore retinal function is limited. There are many similarities in the etiology and progression between the RCS rat and human, RCS rat has been recognized as a classic model of retinitis pigmentosa. Pathological changes of neurons of the RCS rat will provide clues on protection, rescue and rehabilitation of visual function.In recent years, retinal transplantation, visual prosthesis and retinal prosthetic implantation are the main strategies to restore the retinal. We believe that the key to the success of these strategies including: (1)The second-order neurons of receptors hould maintain a stable morphological structure; (2) Donor tissue or cells can form functional synaptic connections with receptors; (3)Choose appropriate therapeutic time window. Results of this study will provide experimental evidence, theoretical basis and new ideas to the selection of RP genes and molecular therapeutic time window, the stability of implanted cells and prosthesis and the integration of therapeutic precursor cells.Based on the research situation, we adopted retinal frozen sections, immunofluorescence staining and DiI trace labeling to study the morphology changes of horizontal cell and related neurons during retinal degeneration. The study included two parts:Part One: Morphology changes of horizontal cells in RCS rat during retinal degenerationPhotoreceptors and the number of ribbon synapses constantly reduced during the process of retinal degeneration in RCS rat, This process can continue until P90d, photoreceptor and ribbon synapse lost slowly. The thickness of ONL in RCS rats was only 15% of control rats at P2M, and at P3M ONL remained only one layer cells. With the progress of retinal degeneration, at P1M, P45d the axon terminals of horizontal cells had no significant changes. At P3M it changed much and extended to ONL. The results from DiI trace labeling showed that the morphology of dendrites and axon terminals of horizontal cells had no changes at P1M, P45d. However, at P2M they changed significantly: Compared with the normal control group,the field of dendrites and axon terminals became small (P<0.05), the small branches of dendrites and axon terminals lost, and the ratio of dendrite length to axon length also shrinked (P<0.05).Part Two: Morphology changes of rod bipolar cells and amacrine cells in RCS rat during retinal degeneration.The results showed the top dendrites of rod-bipolar cell atrophy at P2M; At P3M the dendrites sprouted new process in RCS rat. there were no changes of amacrine cells’dendrites lamination in INL till the late stage of retinal degeneration.Conclusions:1. The morphology changes of horizontal cell and rod bipolar cell in RCS rats are secondary reactions caused by photoreceptor apoptosis, the stabilization of structure is dependent on photoreceptors.2. The morphology changes of horizontal cell and rod bipolar cell in RCS rats did not affect the lamination of amacrine cells’dendrites in IPL.更多还原