Project Summary Glaucoma represents a number of complex diseases with a common endpoint of retinal ganglion cell (RGC) and optic nerve degeneration. Two major models of glaucoma pathogenesis exist – the mechanical hypothesis, which is based on the interaction of intraocular pressure (IOP) and intracranial pressure (ICP), and the vascular hypothesis, which is based on factors that reduce blood flow to RGCs and the optic nerve. Preliminary results from our laboratory suggest that experimental manipulations of mechanical factors such as IOP and ICP in mice result in a range of microvascular and hypoxic abnormalities in the retina. These abnormalities appear to differ not only according IOP and ICP level and exposure duration, but among retinal cell types. In particular, we are interested in RGCs and amacrine cells (ACs), which are critical upstream regulators of RGC function. In this renewal application, we propose to identify the earliest differential responses of RGCs, ACs, and the retinal vasculature to IOP and ICP variation, and to determine the impact of the hypoxic mechanisms that underlie these responses. There are three specific aims: (1) determine the mechanism and differential susceptibilities of retinal capillary plexi to changes in IOP and/or ICP; 2) delineate the differential hypoxic responses that occur in RGCs and ACs after changes in IOP, and test the hypothesis that hypoxia in ACs causes physiologic dysfunction in RGCs; and 3) to test the hypothesis that HIF1, the primary regulator of the hypoxic response, is required for ICP-induced RGC injury. Throughout these Aims, we will employ novel experimental tools that enable us to elevate IOP and ICP to predictable levels for specific durations, which allow us to assess the effects of both magnitude and duration of IOP/ICP change. We will also use a new technique to isolate and culture adult RGCs and AC with high fidelity to probe the differential responses of both cell types to hypoxia and preceding IOP injury. Used in conjunction with a series of in vivo and post mortem electrophysiologic, behavioral, anatomic, and transcriptomic assessments of RGCs, ACs, and the retinal vasculature in both wild type and transgenic mice, we will determine the relative contributions of IOP and ICP change, and assess how alteration of hypoxia and the hypoxic response modifies these contributions to impact RGC/AC dysfunction and survival. Our research will provide an important link between mechanical and vascular hypotheses of glaucoma pathogenesis, potentially identifying a unified theory for susceptibility to glaucoma that can guide future translational diagnostic and therapeutic studies.