Real-time Visualization of AR-13324 Effects on Schlemm’s Canal and Scleral Vessels in Living Mice Guorong Li1, Dibyendu Mukherjee1, Iris Navarro1, Sina Farsiu1,2, Pedro Gonzalez1, Casey C. Kopczynski3 and W. Daniel Stamer1,2 Duke University Eye Center1, Department of Biomedical Engineering2, Aerie Pharmaceuticals, Inc.3 Purpose: The goals of this study were to (i) monitor the effects of a topical rho kinase inhibitor (AR- 13324) on conventional outflow tissues using spectral domain-optical coherence tomography (SD-OCT) at controlled pressure levels in living mouse eyes and (ii) test new software to quantify changes. Methods: Two strains of young mice (C57 and CD1, 2-5 months old) were given AR-13324 or placebo topically. Schlemm’s Canal (SC) was imaged by OCT while intraocular pressure was held at 10, 15 or 30 mmHg. New software was developed to quantitatively assess changes in cross sectional area of SC and scleral vessels. Effects of AR-13324 on fluorescent tracer deposition in anterior segment flat mounts, intraocular pressure (IOP) and outflow facility were also determined. Results: AR-13324 significantly lowered IOP by 5.1±0.5 mmHg, corresponding to an increase in outflow facility of 166% ± 33% in C57 mice. Fluorescence due to tracer deposition in outflow tissues was increased by 2.9-fold in the presence of AR-13324. When holding IOP at 10 mmHg, the average area of SC was 381.8±51.5 µm2, dilating by 132% ± 14%, upon AR-13324 treatment. At elevated IOPs, AR-13324 prevented SC collapse. Moreover, AR-13324 increased the relative speckle variance intensity and total area of scleral vessels conducting aqueous humor by 173% ± 29% and 193% ± 33% respectively. Reproducibility of the software to quantify SC area was 91.7 % ± 7.4 % and 99.7 % ± 4.4 % (inter- and intra-observer). Similar results were observed in CD1 mice. Conclusion: IOP-lowering of AR-13324 in living mice works predominantly by increasing perfusion of conventional outflow tissues. The changes of SC lumen and flow pattern in scleral vessels can be effectively monitored by OCT and discriminated by our newly developed software. This study in living animals is a first step towards the development of OCT technology to monitor glaucoma drug treatment responses in humans.