Project Summary/Abstract Glaucoma is the leading cause of irreversible blindness and is projected to affect 112 million people worldwide by 2040. Women represent 59% of the glaucoma population, highlighting the need to understand sex-specific risk factors for glaucoma. In addition, altered estrogen levels, signaling, or metabolism play a role in glaucoma in both sexes. In women, estrogen receptor polymorphisms are associated with ocular hypertension (an important causal risk factor for glaucoma). Moreover, post-menopausal women have a 1-3 mmHg higher intraocular pressure (IOP) than pre-menopausal women, while hormone therapy containing estrogen reduces IOP. These data suggest that menopause and estrogen levels influence IOP, but the mechanism is unknown. IOP is affected by many factors, but aqueous outflow resistance is the key determinant, which in turn is primarily controlled by trabecular meshwork (TM) function. Intriguingly, outflow resistance correlates with TM stiffness, and it is known that menopause and estrogen levels affect stiffness of many tissues outside the eye, suggesting a possible mechanism by which altered estrogen levels affect IOP. Menopause also alters gene expression, with many genes affected by menopause known to influence aqueous outflow resistance. Thus, it is hypothesized that menopause increases aqueous outflow resistance by stiffening the TM, thus increasing a woman’s risk of developing glaucoma, and that these effects are mediated by altered gene expression profiles in the aqueous outflow pathway. This proposal addresses the hypothesis in an animal model of menopause as follows: Aim 1 will measure IOP and aqueous outflow resistance. Aim 2 will measure TM stiffness using Atomic Force Microscopy. Aim 3 will assess gene expression in aqueous outflow pathway tissues using RNAseq. This proposal achieves these aims using several approaches: (A) It employs a well-established model of menopause, ovariectomy (OVX), in female Brown Norway rats (9-10 months of age); and (B) It will determine if topical estrogen therapy mitigates the effects of OVX. This proposal will also investigate whether topical estrogen therapy affects IOP and aqueous outflow resistance in male rats (9-10 months of age), with an eye towards eventual clinical translation. In all animals, IOP will be measured using rebound tonometry for 8 weeks, followed by measurement of outflow resistance (via iPerfusion) and TM stiffness (via atomic force microscopy) in one eye. Contralateral eyes will be used to assess: (1) estrogen levels, (2) TM structure and composition, or (3) gene expression. Preliminary data support this hypothesis, showing that IOP and aqueous outflow resistance are increased by OVX. This proposal will build on these data and expects to demonstrate that menopause increases TM stiffness and alters gene expression in aqueous outflow pathway tissues, and that topical estrogen therapy prevents these changes. These findings will provide functional and m...