Rho-kinase inhibitor Y-27632 (Y-27) affects actomyosin cytoskeletal networks and has been shown to significantly increase outflow facility (C) in enucleated porcine and rabbit eyes, as well as in vivo monkey eyes without obvious toxicity. The mechanisms underlying these responses remain largely unknown. In this study, we investigate how Y-27 affects aqueous humor C, the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular connective tissue (JCT). 12 bovine eyes were perfused at 15 mmHg with Dulbecco's PBS containing 5.5 mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50 microM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5 microm; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusion-fixed with Karnovsky's fixative. Radial and frontal sections were prepared in all quadrants and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and filtration length (FL) of the IW were measured in > or =16 images/eye, and the average percent effective filtration length (PEFL=FL/TL) was calculated. Sections with AP were processed and examined by light and electron microscopy. The TL of the IW and length exhibiting JCT/IW separation (SL) were measured in > or =16 micrographs/eye, and the average percent separation length (PSL=SL/TL) was also calculated. After Y-27 treatment, C increased from 1.54+/-0.34 (+/-SEM) to 2.36+/-0.54 microL/min per mmHg (58.2+/-18.9%) while control eyes changed from 1.67+/-0.41 to 1.71+/-0.39 microl/min per mmHg (6.0+/-9.3%) and the percent changes between the Y-27-treated and control eyes were significant (p=0.03). Control eyes showed segmental distribution of tracer in the trabecular meshwork tending to cluster near collector channel ostia, whereas Y-27-treated eyes showed a more uniform pattern and extensive labeling along the IW. In Y-27-treated eyes, PEFL was 3-fold larger (57.6+/-3.7%) compared to control eyes (18.2+/-4.5%; p<0.001). Light microscopic examination revealed that, with Y-27, the IW and JCT were significantly distended compared to control eyes, with discernible separation between the IW and JCT. PSL was 2.8-fold larger in Y-27-treated eyes (59.3+/-3.6%) than in controls (20.8+/-2.0%; p<0.001). A significant positive correlation was found between PEFL and PSL (p=0.003) suggesting that as connectivity between the JCT and IW decreases the available area for aqueous humor drainage increases along the AP. Our study also demonstrates a significant positive correlation between C and the PSL (p=0.01), a finding identical to what we reported to occur during the "washout" effect in bovine eyes. Our data suggests the structural correlate to the increase in C and PEFL after Y-27-treatment is physical separation between the JCT and IW. The increase in C after Y-27-treatment may share a similar mechanism comparable with the washout effect that occurs in bovine eyes. Overall, these findings support our hypothesis that JCT/IW connectivity influences local outflow hydrodynamics that regulate C, and suggest that strategies targeting JCT/IW connectivity are promising anti-glaucoma therapies to reduce IOP.