Glaucoma, Neurotrophins, and the Lamina Cribrosa

青光眼、神经营养素和筛板

• 批准号: 6621728
• 负责人: ROBERT J WORDINGER
• 金额: $ 28.4万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-03-01 至 2005-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621728
• 关键词: astrocytes; axon; cell line; cell morphology; cell proliferation; clinical research; enzyme activity; enzyme linked immunosorbent assay; glaucoma; human tissue; immunocytochemistry; immunologic assay /test; intraocular pressure; messenger RNA; neurotrophic factors; optic nerve; paracrine; polymerase chain reaction; protein tyrosine kinase; receptor expression; retinal ganglion; tumor necrosis factor alpha; visual fields; western blottings

In primary open-angle glaucoma, the major site of damage is within the lamina cribrosa (LC) region of the optic nerve head (ONH). The LC consists of a connective tissue framework through which millions of unmyelinated retinal ganglion cell axons exit the eye and form the optic nerve. Non-neural cells within the LC are closely associated with individual axons and are know to synthesize ECM components. Neurotrophins (NT) include NGF, BDNF, NT-3, and NT-4 and are known to signal via high affinity Trk receptors. While originally thought to only regulate neuronal survival, non-neuronal cells have recently been shown to express NT and signal via Trk receptors. This raises the possibility that non-neuronal cells within the human LC may synthesize and secrete NT locally to control the microenvironment and/or maintain the viability of RGC. Our long-term research objective is to increase our knowledge of the physiological roles of NT within the normal and glaucomatous human LC. The hypothesis of this proposal is that NT expression and paracrine signaling within the human LC is decreased in glaucoma and under experimental glaucomatous conditions. The following specific aims will be used to test the hypothesis: (1) isolate and characterize non-neural cells from normal and glaucomatous human LC tissues, (2) compare and contrast NT and Trk receptor mRNA and protein expression in cells isolated from the LC of normal donors, (3) demonstrate that ONH astrocytes and LC cells secrete and respond to NT, (4) compare and contrast NT and Trk receptor mRNA and protein expression and NT secretion in cells from glaucomatous donors, (5) compare and contrast functional responses (e.g. cell proliferation and ECM modulator expression) to exogenous NT in cells from normal and glaucomatous donors, and (6) using ischemic and hypoxic conditions, compare and contrast NT and Trk receptor expression, NT secretion, and functional responses in ONH cells from normal and glaucomatous donors. To achieve the specific aims we will use RT-PCR to determine gene expression, Real-time Quantitative RT-PCR to determine mRNA levels, cell proliferation assays, Western-blotting and immunohistochemistry to demonstrate protein expression, ELISA immunoassay to determine NT, MMP and TIMP secretion and phosphorylation assays to demonstrate Trk activation. This study is significant because it will increase our knowledge of the role of NT in the human LC and may lead to alternative treatment modalities in glaucoma.

在原发性开角青光眼中，损伤的主要部位位于视神经头（ONH）的椎板cribrosa（LC）区域内。 LC由一个结缔组织框架组成，通过该框架，成千上万的无髓神经节细胞轴突将眼睛排出并形成视神经。 LC内的非神经细胞与单个轴突密切相关，并且知道可以合成ECM成分。 神经营养蛋白（NT）包括NGF，BDNF，NT-3和NT-4，已知通过高亲和力TRK受体发出信号。 虽然最初被认为仅调节神经元存活，但最近已显示非神经元细胞通过TRK受体表达NT和信号。 这增加了人类LC内的非神经元细胞可以合成并在本地分泌以控制微环境和/或维持RGC的生存能力的可能性。 我们的长期研究目标是提高我们对NT在正常和青光眼人类LC中的生理作用的了解。 该提议的假设是，在青光眼和实验性青光眼条件下，人LC内的NT表达和旁分泌信号传导降低。 以下具体目的将用于检验假设：（1）分离和表征来自正常和青光眼人体LC组织的非神经细胞，（2）比较和对比NT和TRK受体mRNA和TRK受体mRNA以及从LC中分离的细胞中的蛋白质表达（3）表明，ONH星形胶质细胞和LC细胞分泌并响应NT，（4）比较和对比NT和TRK受体mRNA以及蛋白质表达以及蛋白质的表达以及蛋白质的蛋白质和NT分泌，（5）比较和对比功能函数反应（例如细胞增殖和ECM调节剂表达）对来自正常和青光眼供体细胞中外源性NT的反应，以及（6）使用缺血性和低氧条件，比较和对比NT和TRK受体表达，NT分泌，NT分泌和功能反应，以及来自ONH细胞的功能反应正常和青光眼供体。 为了达到具体目标，我们将使用RT-PCR确定基因表达，实时定量RT-PCR确定mRNA水平，细胞增殖分析，西部印象和免疫组织化学来证明蛋白质表达，ELISA免疫测定以确定NT，MMP和MMP和MMP和MMP和MMP和TIMP分泌和磷酸化测定法证明了TRK激活。 这项研究很重要，因为它将增加我们对NT在人类LC中的作用的了解，并可能导致青光眼的替代治疗方式。