In vivo trabecular meshwork gene expression response to elevated IOP

项目摘要 眼内压升高是青光眼的主要危险因素，降低眼内压是唯一有效的方法。 减缓青光眼视力丧失的临床策略。眼压由小梁网（TM）调节， 建立了对房水流出的阻力。先前使用离体器官培养模型的研究表明， 6 - 8小时持续的压力升高诱导IOP稳态，其中阻力被重塑， 促进房水流出并减轻IOP。这涉及许多上调和下调的基因。IOP时 降低，这些"稳态"基因理论上应该在24 - 48小时内恢复到正常表达水平， 但尚未对此进行调查。这些事件仅在离体器官培养中进行了研究， 缺乏正常的昼夜IOP波动、巩膜外静脉压和持续的房水形成。更好 了解这种稳态反应需要研究体内TM基因表达的变化， 控制眼压挑战。存在各种体内啮齿动物IOP模型，但是用于产生IOP的技术方法不多见。 压力升高损害TM细胞功能并导致IOP的不可预测的峰值。受控 眼压升高（CEI）大鼠模型是研究这些体内TM基因表达变化的理想模型。在这里， 放置在虹膜前面的前房中，并输送无菌平衡盐溶液以升高 压力因此，可以对眼睛施加已知持续时间的限定的IOP升高。重要的是，由于角度 当眼压升高时，眼压升高会产生TM的拉伸/扭曲，类似于青光眼患者中发现的情况。这 这使我们首次能够研究TM中体内IOP相关基因表达的变化。在这项研究中，我们将 使用RNA-seq鉴定在响应于单次IOP暴露和从单次IOP暴露恢复时改变的TM基因。我们 假设体内CEI后TM基因表达的变化将鉴定与以下相关的新途径： 眼压稳态。在SA#1中，将在眼睛承受50 mmHg压力升高的情况下进行CEI 8小时（CEI 50）。对照将包括暴露于20 mmHg 8小时（CEI 20）的动物眼睛， 未经麻醉或手术操作的幼稚动物。CEI后立即（0小时），将RNA 将从TM组织分离并进行RNA-seq。生物统计学分析将显著提高- 并下调体内IOP相关基因。在SA#2中，将进行CEI 50和CEI 20，但大鼠将 恢复24或48小时。RNA-seq样本将与SA#1至SA#2的样本同时运行。 允许我们在所有时间点（0、24、48小时）严格比较CEI 50和对照组。这些分析 将使我们能够将IOP稳态基因（那些在24 - 48小时内恢复的基因）与非稳态基因分开， 基因（表现出延长反应的基因）。总之，这项研究的结果将提供一个全面的 鉴定活体眼内IOP稳态的TM基因谱。我们希望发现新的基因， 控制体内IOP稳态的途径，这将有助于设计新的治疗方法， 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.