In vivo trabecular meshwork gene expression response to elevated IOP

项目摘要 高眼压是青光眼的主要危险因素，降低眼压是唯一有效的方法。 延缓青光眼视力丧失的临床策略。眼压由小梁网络(TM)调节，它 建立对房水流出的抵抗力。之前使用体外器官培养模型的研究表明 持续6-8小时的压力升高会引起眼压的动态平衡，此时眼压的阻力被重新调整为 促进房水流出，减轻眼压。这涉及到许多上调和下调的基因。根据IOP 从理论上讲，降低这些“内稳态”基因应该会在24-48小时内恢复到正常的表达水平， 但这一点尚未得到调查。这些事件只在体外器官培养中被研究过， 缺乏正常的昼夜眼压波动、巩膜外静脉压和持续的房水形成。更好 要了解这种稳态反应，需要研究体内TM基因表达的变化。 控制眼压的挑战。活体啮齿动物眼压模型有多种，但用于创建 压力升高会损害TM细胞的功能，并导致不可预测的眼压峰值。受控者 眼压升高(CEI)大鼠模型是研究体内TM基因表达变化的理想模型。在这里，插管是 放置在前房，虹膜前方，并输送无菌平衡盐溶液以抬高 压力。因此，可以将已知持续时间的已定义的眼压升高应用于眼睛。重要的是，由于角度 如果眼压升高是开放的，则会导致青光眼患者的TM出现拉伸/变形。这 使我们第一次能够在体内研究TM中IOP相关基因表达的变化。在这项研究中，我们将 使用RNA-seq来识别因单次眼压暴露而改变的TM基因，并从中恢复。我们 假设TM基因在体内CEI后表达发生变化，将发现与以下相关的新途径 眼压动态平衡。在SA#1中，将在眼压升高50毫米汞的情况下进行CEI 持续8小时(CEI 50)。控制措施将包括暴露于20毫米汞8小时的动物眼睛(CEI 20)和 幼稚的动物，没有麻醉剂或外科手术。在CEI之后(0小时)，RNA将被 从TM组织中分离并进行RNA-SEQ。生物统计分析将确定显著上升- 并下调体内眼压相关基因的表达。在SA#2中，将执行CEI 50和CEI 20，但大鼠将 允许恢复24或48小时。RNA-seq样品将与SA#1至 允许我们严格比较CEI50和对照组在所有时间点(0、24、48小时)。这些分析 将使我们能够将眼压稳态基因(那些在24-48小时内恢复的基因)从非稳态中分离出来 基因(表现出长时间反应的基因)。总而言之，这项研究的结果将提供一个全面的 活眼眼压动态平衡TM基因图谱的鉴定我们希望发现新的基因或 体内控制眼压稳态的途径，这将有助于设计针对增强眼压的新疗法 青光眼患者眼压控制的内源性机制。

Project Summary Elevated intraocular pressure (IOP) is a primary risk factor for glaucoma and lowering IOP is the only effective clinical strategy to slow glaucomatous vision loss. IOP is regulated by the trabecular meshwork (TM), which builds a resistance to aqueous humor outflow. Previous studies using an ex vivo organ culture model showed that a 6-8 hour sustained pressure elevation induces IOP homeostasis, where the resistance is remodeled to facilitate aqueous outflow and alleviate IOP. This involves many up- and down-regulated genes. Upon IOP lowering, these `homeostatic' genes should theoretically recover to normal expression levels within 24-48 hours, but this has not yet been investigated. These events have only been studied in ex vivo organ cultures, which lack a normal diurnal IOP fluctuation, episcleral venous pressure and ongoing aqueous humor formation. Better understanding of this homeostatic response requires studying in vivo TM gene expression changes in response to a controlled IOP challenge. Various in vivo rodent IOP models exist, but the technical methods used to create pressure elevation compromises TM cell function and causes unpredictable spikes in IOP. The Controlled Elevation of IOP (CEI) rat model is ideal to study these in vivo TM gene expression changes. Here, a cannula is placed into the anterior chamber, anterior to the iris, and sterile balance salt solution is delivered to elevate pressure. A defined IOP elevation of known duration can thus be applied to the eye. Importantly, since the angle is open, IOP elevation produces stretching/distortion of the TM mimicking that found in glaucoma patients. This allows us, for the first time, to study in vivo IOP-related gene expression changes in the TM. In this study, we will use RNA-seq to identify TM genes altered in response to, and recovery from, a single IOP exposure. We hypothesize that TM gene expression changes following the in vivo CEI will identify novel pathways related to IOP homeostasis. In SA#1, CEI will be performed where eyes are subjected to a 50 mmHg pressure elevation for 8 hours (CEI 50). Controls will include eyes from animals exposed to 20 mmHg for 8 hours (CEI 20) and naïve animals, without anesthetic or surgical manipulations. Immediately following CEI (0 hours), RNA will be isolated from TM tissue and RNA-seq will be performed. Biostatistical analyses will determine significantly up- and down-regulated IOP-related genes in vivo. In SA#2, CEI 50 and CEI 20 will be performed, but rats will be allowed to recover for 24 or 48 hours. RNA-seq samples will be run concomitant with samples from SA#1 to allow us to rigorously compare CEI 50 and control groups at all time points (0, 24, 48 hours). These analyses will enable us to separate IOP homeostatic genes (those that recover in 24-48 hours) from non-homeostatic genes (genes that display a prolonged response). Together, results from this study will provide a comprehensive identification of the TM gene profile of IOP homeostasis in the living eye. We expect to uncover new genes or pathways that govern IOP homeostasis in vivo, which will help design new therapies directed at augmenting endogenous mechanisms of IOP control in glaucoma patients.