PROJECT SUMMARY/ABSTRACT Glucocorticoids are frequently used to treat inflammatory and autoimmune diseases. Although very effective, glucocorticoids may induce ocular hypertension (OHT)/glaucoma in some patients (they are called responders; the others are called non-responders) after prolonged topical or systemic use. The trabecular meshwork (TM) outflow pathway contains several tissues. From upstream to downstream of aqueous humor outflow, the tissues can be divided into proximal outflow tissues including the TM and Schlemm’s canal, as well as distal outflow tissues including the collector channel and aqueous vein. The importance of the distal flow tissue is not clear in glucocorticoid-induced OHT/glaucoma. Therefore, we will study our hypothesis that the distal outflow tissue, like the TM, also responds differentially to glucocorticoids and this response contributes to OHT. Our goal is to elucidate the molecular mechanism of glucocorticoid-induced OHT. We propose two specific aims. SA1. Determine if distal outflow tissues differentially regulate outflow resistance in GIG. Using our new perfusion culture model, we will perfuse paired human donor corneal rims in two study groups. Group a: Both rims will have the TM removed using the Kahook dual blade. One rim will be perfused with 0.1% ethanol (EtOH) as a vehicle control and the fellow rim with 100nM dexamethasone (DEX) for 7-10 days. IOP will be recorded and compared between eyes. At least 50 pairs of corneal rims will be studied. This group is used to determine if the distal outflow tissue is able to cause glucocorticoid-induced OHT. Group b: One rim will be perfused directly, while the fellow rim will have the TM tissue removed and then perfused. Both rims will be treated with DEX for 7-10 days and IOP will be recorded. At least 50 pairs of corneal rims will be studied. This group is used to determine if the distal outflow tissue is as important as the proximal tissue in glucocorticoid- induced OHT. SA2. Compare spatial gene expression between proximal and distal outflow tissues in GIG. Paired corneal rims with or without TM will be perfused with EtOH in one rim and DEX in the fellow rim. Both rims will be used for spatial gene expression studies. Tissues will be fixed and stained with hematoxylin and eosin to identify the outflow structure/region. Identified regions will be mounted on a chip on a slide. The chip contains 5000 bar-coded spots containing oligonucleotides for the capture, amplification, and identification of tissue RNA for RNA sequencing. The resolution of the chip is sufficient to study both proximal and distal outflow tissues. Both responder and non-responder tissues will be studied and compared. The results will be validated using in situ hybridization and immunostaining. Our project is novel because we will use the new corneal rim perfusion culture model and spatial gene expression technology. This study is significant because it will provide a novel treatment target, the di...