Cell Biology of Astrocytes in Optic Nerve Head

项目摘要 青光眼的典型特征是视网膜神经节细胞的进行性丧失。药物降低 眼内压是青光眼药物治疗的主要手段，但效果不佳 在所有情况下。因此，新的治疗方法将受到欢迎。 许多证据表明，视神经头是神经节细胞轴突的初始损伤点， 青光眼在这个解剖位置，星形胶质细胞的网状结构形成了细胞的直接细胞环境。 轴突视神经受到损伤后，视神经乳头中的星形胶质细胞变得具有反应性。 我们研究了视神经挤压伤后视神经乳头星形胶质细胞反应性的时间进程 形态学和基因表达水平。我们还研究了星形胶质细胞的形态， DBA/2 J小鼠系自发发生青光眼。DBA/2 J小鼠与一种品系杂交， 在单个星形胶质细胞中表达GFP，从而对反应性星形胶质细胞进行显微镜观察 容易.我们的发现之一是，在神经节细胞变性的任何迹象变得明显之前， 在视网膜中，一些星形胶质细胞长出新的纵向突起进入板后轴突束。我们 现在我们将研究驱动这些过程生长的机制及其功能。 我们相信星形胶质细胞的反应性，至少在其早期阶段，是一种有益的反应， 来保护神经节细胞及其轴突青光眼的一种可能的治疗方法是 增强早期星形胶质细胞反应。因此，我们建议研究 控制早期星形胶质细胞反应的视神经。我们比较了不同调控的基因 在我们自己的微阵列筛选中（使用视神经挤压）， 其他实验室使用DBA/2 J小鼠或巩膜外静脉注射高眼压模型， 大鼠我们鉴定了在DBA/2 J中早期上调的信号分子和转录因子， 因此似乎参与调节星形胶质细胞的反应性， 青光眼和外伤后。这些基因中的大多数也在巩膜外静脉中上调 注入模型我们的候选者是信号分子骨形态发生蛋白1和2， 白血病抑制因子）、分泌型磷蛋白1（骨桥蛋白）、脂质运载蛋白2和转化型 生长因子β 1;和转录因子Tcf 19、TGFβ诱导因子同源框1、Runt- 相关转录因子1和2，以及E2 f8。我们将研究他们在三种模式中的参与， 青光眼和视神经挤压后。为此，我们将利用方法上的进步 在第一次授权期间，即通过AAV 2/9有效转染视神经头星形胶质细胞， 分析来自视神经乳头的形态保存良好的解离星形胶质细胞的能力， 以及以“活高尔基体染色”的方式在星形胶质细胞中表达GFP的小鼠品系。

Project summary The characteristic feature of glaucoma is a progressive loss of retinal ganglion cells. Drugs that lower the intraocular pressure are the mainstay of pharmaceutical therapy of glaucoma, but they are not effective in all cases. New therapeutic approaches would therefore be welcome. Much evidence points to the optic nerve head as the point of initial insult to ganglion cell axons in glaucoma. In this anatomical location, a meshwork of astrocytes forms the direct cellular environment of the axons. Following an insult to the optic nerve, the astrocytes in the optic nerve head become reactive. We have studied the time course of astrocyte reactivity in the optic nerve head after nerve crush morphologically and on the level of gene expression. We also studied astrocyte morphology in the DBA/2J mouse line that develops glaucoma spontaneously. DBA/2J mice were crossed with a strain that expresses GFP in individual astrocytes, thus making the microscopic observation of reactive astrocytes easy. One of our findings was that, before any signs of ganglion cell degeneration become obvious in the retina, some astrocytes grow new, longitudinal processes into the retrolaminar axon bundles. We are now going to study the mechanisms that drive the growth of these processes and their function. We believe that astrocyte reactivity, at least in its early phase, is a beneficial response that aims to protect ganglion cells and their axons. A possible therapeutic approach to glaucoma would be to enhance the early astrocytic response. We therefore propose to study the regulatory mechanisms in the optic nerve that govern early astrocyte reactivity. We compared the genes that are differentially regulated in our own microarray screen (using optic nerve crush) with those that were reported in recent studies from other laboratories using DBA/2J mice or the episcleral vein injection model of ocular hypertension in rats. We identified signaling molecules and transcription factors that were up-regulated early in DBA/2J mice and after nerve crush and therefore appear to be involved in regulating astrocyte reactivity both in glaucoma and after traumatic injury. Most of these genes are also up-regulated in the episcleral vein injection model. Our candidates are the signaling molecules Bone Morphogenetic Proteins 1 and 2, Leukemia Inhibitory Factor), Secreted Phosphoprotein 1 (osteopontin), Lipocalin 2, and Transforming Growth Factor beta 1; and the transcription factors Tcf19, TGFβ-Induced Factor Homeobox 1, Runt- Related Transcription factors 1 and 2, and E2f8. We will study their involvement in three models of glaucoma, and after optic nerve crush. For this purpose, we will make use of methodological advances during the first grant period, namely the efficient transfection of optic nerve head astrocytes by AAV2/9, the ability to analyze dissociated astrocytes with well-preserved morphology from the optic nerve head, and a mouse strain that expresses GFP in astrocytes in the manner of a “live Golgi stain”.