In vivo trabecular meshwork gene expression response to elevated IOP

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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.