Project Summary Understanding cellular mechanisms of intraocular pressure (IOP)-induced axonal injury will help develop new glaucoma treatments that protect the optic nerve. Our Controlled Elevation of IOP (CEI) model produces optic nerve head (ONH) gene expression changes and optic nerve (ON) damage that parallel those observed in chronic models. An RNAseq time-course analysis has revealed that IOP produces early activation of several major pathways and specific recovery of their components. We have now developed a method of creating awake CEI (aCEI) exposures in conscious rats using an indwelling anterior chamber cannula that is externally accessible. This will allow us to study events of chronic glaucoma, which occurs over years, in the relatively short time frame of a laboratory setting. In Specific Aim 1, we will (a) demonstrate that an 8-hour aCEI at 40 mmHg will produce little to no injury compared to 50 mmHg, (b) show that a non-injurious, lower IOP will produce ONH gene expression changes that are less than, but qualitatively similar, to those seen with an injurious, higher IOP and (c) show that exposure to aCEI in elderly animals will produce greater injury than the same level of pressure in adult animals. Specific Aim 2 will show that (a) the timing of a second, injurious aCEI following an initial, similar exposure will affect additivity of the axonal injury and (b) the timing of a repeat, injurious aCEI will affect ONH gene expression and recovery. Specific Aim 3 will demonstrate that (a) repeat aCEI can be used to model “chronic” glaucomatous optic nerve damage, (b) an eye with pressure-induced optic nerve injury will be more susceptible to subsequent IOP exposure than an eye without prior injury and (c) IOP fluctuations produce more injury than the same level of IOP maintained at a steady state for the same duration. These studies in unanesthetized animals use levels of IOP that, relative to normal, mean rat IOP, are comparable to human glaucoma, and will provide the most accurate representation of this aspect of the human disease possible in laboratory rats. They will provide unique insights into cellular mechanisms of chronic glaucomatous optic nerve damage and allow study of previously unapproachable aspects of this chronic disease.