The Mechanism Of Increasing Outflow Facility By Rho-Kinase Inhibition With Y27632

The Mechanism of Increasing Outflow Facility by Rho-Kinase Inhibition with Y27632 in Bovine EyesPurpose: To investigate how Y27632 (Y27; a selective inhibitor of Rho-kinase and cell contractility) affects outflow facility (C), the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular tissue (JCT) in bovine eyes.Methods: Twelve bovine eyes were perfused at 15mmHg with Dulbecco's PBS containing 5.5 mM glucose (GPBS) to establish a stable baseline facility. The anterior chamber contents were exchanged for GPBS with 50μM Y27 in 7 eyes, while 5 eyes received GPBS alone. Eyes were perfused with or without Y27 (0.5 ml), followed by an exchange with GPBS containing fluorescent microspheres (0.5μm; 0.002% v/v) and subsequent perfusion at a fixed volume (0.5 ml) to deliver equivalent amounts of tracer that label the hydrodynamic patterns of outflow. Eyes were then perfusion-fixed with Karnovsky's fixative. Radial sections and sections tangential to the corneoscleral limbus and perpendicular to the ocular surface in all quadrants were prepared and confocal images were taken along the IW of the aqueous plexus (AP). The total length (TL) and the tracer-decorated length (L) of IW were measured in >16 images/eye, and the average percent effective filtration length (PEFL=L/TL) was calculated for each eye. The sections with AP were then processed and examined under light and electron microscopy. The TL of IW and the length exhibiting IW and JCT separation (SL) were measured in >16 micrographs/eye, and the average percentage separation length (PSL= SL/TL) was also calculated. Results: After Y27 treatment, the increase of outflow facility was 0.83±0.26 ul/min/mmHg while the change of outflow facility was 0.04±0.14 ul/min/mmHg in sham-treated control eyes (p=0.03) and the percent changes between the Y27 treated eyes (58.2±18.9%) and control eyes (6.0±9.3%) were significant (p=0.04). Control eyes showed segmental distribution of tracer in the trabecular meshwork (TM), with clustering of microspheres near collector channel ostia. Y27 treated eyes showed a more uniform pattern, with extensive labeling along the IW. The PEFL in Y27 treated eyes (57.6±3.7%) was 3-fold larger than that in control eyes (18.2±4.5%, p<0.001). Light microscopic examination revealed that with Y27 the IW and JCT were significantly distended compared to control eyes, with discernable separation between the IW and JCT. The percentage separation length (PSL) was 2.8-fold larger in Y27 treated eyes (59.3±3.6%) than that in control eyes (20.8±2.0%, P<0.001).Conclusions: Y27 significantly increases outflow facility, the PEFL and the PSL along the IW of the AP in bovine eyes. The changes in outflow facility and PSL are consistent with previous findings that correlate with increasing outflow facility during "washout". Therefore Y27 and washout may act through similar mechanisms that increase outflow facility by redistributing aqueous outflow through a larger area of the IW and JCT. These findings support the hypothesis that inner wall/JCT connectivity influences local outflow hydrodynamics to control outflow facility. The Mechanism of Increasing Outflow Facility by Rho-Kinase Inhibition with Y27632 in Monkey EyesPurpose: To investigate how Y27632 (Y27; a selective inhibitor of Rho-kinase and cell contractility) affects outflow facility (C), the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular tissue (JCT) in monkey eyes.Methods: Eight monkey eyes were perfused at 15mmHg with Dulbecco's PBS containing 5.5 mM glucose (GPBS) to establish a stable baseline facility. The anterior chamber contents were exchanged for GPBS with 50μM Y27 in 4 eyes, while 4 eyes received GPBS alone. Eyes were perfused with or without Y27 (0.3 ml), followed by an exchange with GPBS containing fluorescent microspheres (0.5μm; 0.002% v/v) and subsequent perfusion at a fixed volume (0.3 ml) to deliver equivalent amounts of tracer that label the hydrodynamic patterns of outflow. Eyes were then perfusion-fixed with Karnovsky's fixative. Radial sections and sections tangential to the corneoscleral limbus and perpendicular to the ocular surface in all quadrants were prepared and confocal images were taken along the IW of the Schlemm's canal (SC). The total length (TL) and the tracer-decorated length (L) of IW were measured in >16 images/eye, and the average percent effective filtration length (PEFL=L/TL) was calculated for each eye. The sections with SC were then processed and examined under light and electron microscopy. The TL of IW and the length exhibiting IW and JCT separation (SL) were measured in >16 micrographs/eye, and the average percentage separation length (PSL= SL/TL) was also calculated.Results: After Y27 treatment, the increase of outflow facility was 0.59±0.11μl/min/mmHg while the change of outflow facility was 0.18±0.05μl/min/mmHg in sham-treated control eyes (p=0.03) and the percent changes between the Y27 treated eyes (114.9±14.9%) and control eyes (44.3±9.7%) were significant (p=0.01). Control eyes showed segmental distribution of tracer in the trabecular meshwork (TM), with clustering of microspheres near collector channel ostia. Y27 treated eyes showed a more uniform pattern, with extensive labeling along the IW. The PEFL in Y27 treated eyes (82.5±1.2%) was 3.4-fold larger than that in control eyes (24.2±4.2%, p<0.001). Light microscopic examination revealed that with Y27 the IW and JCT were significantly distended compared to control eyes, with discernable separation between the IW and JCT. The percentage separation length (PSL) was 2.2-fold larger in Y27 treated eyes (75.2±4.0%) than that in control eyes (33.5±5.3%; p=0.001).Conclusions: Y27 significantly increases outflow facility, the PEFL and the PSL along the IW of the SC in monkey eyes. The changes in outflow facility and PSL are consistent with previous findings that correlate with increasing outflow facility during "washout". Therefore Y27 and washout may act through similar mechanisms that increase outflow facility by redistributing aqueous outflow through a larger area of the IW and JCT. These findings support the hypothesis that inner wall/JCT connectivity influences local outflow hydrodynamics to control outflow facility. The Mechanism of Increasing Outflow Facility by Rho-Kinase Inhibition with Y27632 in Human EyesPurpose: To investigate how Y27632 (Y27; a selective inhibitor of Rho-kinase and cell contractility) affects outflow facility (C), the hydrodynamic patterns of outflow, and the morphology of the inner wall (IW) and juxtacanalicular tissue (JCT) in human eyes.Methods: Eight human eyes were perfused at 15 mmHg with Dulbecco's PBS containing 5.5 mM glucose (GPBS) to establish a stable baseline facility. The anterior chamber contents were exchanged for GPBS with 50μM Y27 in 4 eyes, while 4 eyes received GPBS alone. Eyes were perfused with or without Y27 (0.3 ml), followed by an exchange with GPBS containing fluorescent microspheres (0.5μm; 0.002% v/v) and subsequent perfusion at a fixed volume (0.3 ml) to deliver equivalent amounts of tracer that label the hydrodynamic patterns of outflow. Eyes were then perfusion-fixed with Karnovsky's fixative. Radial sections and sections tangential to the corneoscleral limbus and perpendicular to the ocular surface in all quadrants were prepared and confocal images were taken along the IW of the Schlemm's canal (SC). The total length (TL) and the tracer-decorated length (L) of IW were measured in >16 images/eye, and the average percent effective filtration length (PEFL=L/TL) was calculated for each eye. The sections with SC were then processed and examined under light and electron microscopy.Results: After Y27 treatment, the change of outflow facility was 0.02±0.01μl/min/mmHg while the change of outflow facility was 0.02±0.03μl/min/mmHg in sham-treated control eyes (p=0.98) and the percent changes between the Y27 treated eyes (8.1±3.0%) and control eyes (11.3±13.4%) were also no significant difference (p=0.83). Control eyes and Y27 treated eyes both showed segmental distribution of tracer in the trabecular meshwork (TM), with clustering of microspheres near collector channel ostia. The PEFL in Y27-treated eyes was 47.4±6.5% and 39.0±12.5% in control eyes and no significant difference between 2 groups (p=0.41). Light microscope and electron microscope examination revealed that the IW and JCT were contact and without discernable separation between the IW and JCT both in Y27 treated eyes and in control eyes. Electron microscopy revealed that large amount of elastic fibers were found under the inner wall of Schlemm's canal and in JCT and trabecular meshwork.Conclusions: Y27 can not increases outflow facility and the PEFL in human eyes. Y27can not induce separation between inner wall of Schlemm's canal and JCT. This may be resulted from strengthened connection between IW and JCT in human eyes. These findings also support the hypothesis that inner wall/JCT connectivity influences local outflow hydrodynamics to control outflow facility.