The mechanism of increasing outflow facility by rho-kinase inhibition with Y-27632 in bovine eyes

• Published in: Experimental eye research 2008-02, Vol.86 (2), p.271-281
• Main Authors: Lu, Zhaozeng, Overby, Darryl R., Scott, Patrick A., Freddo, Thomas F., Gong, Haiyan
• Format: Article
• Language: English
• Subjects: Amides - pharmacology; Animals; Aqueous Humor - drug effects; Aqueous Humor - metabolism; aqueous outflow facility; bovine eye; Cattle; confocal microscopy; electron microscopy; Enzyme Inhibitors - pharmacology; inner wall; JCT; light microscopy; Microscopy, Confocal; Microscopy, Electron; Microspheres; Organ Culture Techniques; Pyridines - pharmacology; Rheology; rho-Associated Kinases - antagonists & inhibitors; Sclera - drug effects; Sclera - ultrastructure; trabecular meshwork; Trabecular Meshwork - diagnostic imaging; Trabecular Meshwork - drug effects; Ultrasonography; Vacuoles - drug effects; washout; Y-27632
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1016/j.exer.2007.10.018

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 15mmHg with Dulbecco's PBS containing 5.5mM glucose (DPBS) to establish stable baseline C. The anterior chamber was exchanged and perfused with DPBS containing 50μM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5μm; 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 ≥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 ≥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±0.54μL/min per mmHg (58.2±18.9%) while control eyes changed from 1.67±0.41 to 1.71±0.39μl/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