Glaucoma, Neurotrophins, and the Lamina Cribrosa

青光眼、神经营养素和筛板

• 批准号: 6730406
• 负责人: ROBERT J WORDINGER
• 金额: $ 4.53万
• 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/6730406
• 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.

在原发性开角型青光眼中，主要损伤部位位于视神经乳头 (ONH) 的筛板 (LC) 区域内。 LC 由结缔组织框架组成，数以百万计的无髓鞘视网膜神经节细胞轴突通过该框架离开眼睛并形成视神经。 LC 内的非神经细胞与单个轴突密切相关，并且已知可以合成 ECM 成分。 神经营养素 (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，但最近显示非神经元细胞可以通过 Trk 受体表达 NT 和信号。 这提出了人类 LC 内的非神经元细胞可能在局部合成和分泌 NT 以控制微环境和/或维持 RGC 活力的可能性。 我们的长期研究目标是增加我们对 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，人类 LC 内的 NT 表达和旁分泌信号传导减少。 以下具体目标将用于检验该假设：(1) 从正常和青光眼人类 LC 组织中分离和表征非神经细胞，(2) 比较和对比从 LC 中分离的细胞中的 NT 和 Trk 受体 mRNA 和蛋白表达正常供体的细胞，(3) 证明 ONH 星形胶质细胞和 LC 细胞分泌 NT 并对其作出反应，(4) 比较和对比青光眼供体细胞中的 NT 和 Trk 受体 mRNA 和蛋白质表达以及 NT 分泌， (5)比较和对比来自正常和青光眼供体的细胞中对外源NT的功能反应(例如细胞增殖和ECM调节剂表达)，以及(6)使用缺血和缺氧条件，比较和对比NT和Trk受体表达、NT分泌，以及来自正常和青光眼供体的 ONH 细胞的功能反应。 为了实现特定目标，我们将使用 RT-PCR 测定基因表达、实时定量 RT-PCR 测定 mRNA 水平、细胞增殖测定、蛋白质印迹和免疫组织化学测定蛋白质表达、ELISA 免疫测定测定 NT、MMP 和TIMP 分泌和磷酸化检测可证明 Trk 激活。 这项研究意义重大，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。