Similar hydrodynamic and morphological changes in the aqueous humor outflow pathway after washout and Y27632 treatment in monkey eyes

Abstract

Our previous studies in bovine eyes demonstrated that the structural correlate to the increase in outflow facility after either Rho-kinase inhibitor Y-27632 (Y27) treatment or washout appeared to be separation between the juxtacanalicular tissue (JCT) and inner wall (IW) of the aqueous plexus, the bovine equivalent of Schlemm's canal (SC). While these findings suggest that Y27 and washout may increase outflow facility through a similar mechanism, the anatomy of bovine outflow pathway differs considerably from both the human and monkey outflow pathway; however, only the human eye does not exhibit washout. In light of this, we compared the effects of Y27 and washout on outflow facility, hydrodynamic patterns of outflow, and the morphology of the IW and JCT in monkey eyes, given that their anatomy is closer to human eyes. Twelve freshly enucleated monkey eyes were used in this study. Eyes were perfused with Dulbecco's PBS containing 5.5 mM glucose (GPBS) to establish a baseline facility at 15 mmHg. Four eyes were perfused for a short-duration (30 min) as a control, 4 eyes for a long-duration (180 min) to induce washout, and 4 eyes with GPBS+50 μM Y27 for 30 min. All eyes were then perfused with fluorescent microspheres (0.5 μm; 0.002%) to label the hydrodynamic patterns of outflow and then perfusion-fixed. Confocal images of frontal sections were taken along the IW of SC. The total length (TL) and the tracer-decorated length (FL) of the IW were measured to calculate the average percent effective filtration length (PEFL = FL/TL). Sections with SC were examined by light and electron microscopy. The TL of the IW and the length exhibiting separation (SL) in the JCT were measured to calculate the average percent separation length (PSL = SL/TL). Outflow facility increased 149.2% (p < 0.01) from baseline after washout during long-duration perfusion, and 114.9% (p = 0.004) after Y27 treatment, but did not change significantly after short-duration perfusion in control eyes (p = 0.46). Distribution of the tracer labeling appeared punctate along the IW of control eyes, while a more uniform pattern was observed after washout and Y27 treatment. PEFL in washout (83.4 ± 2.1%) and Y27 treated eyes (82.5 ± 1.6%) was 3.4-fold larger compared to controls (24.2 ± 4.2%, P < 0.001). The JCT appeared distended with loss of connections between JCT cells and between JCT cells and their extracelluar matrix in eyes with washout or after Y-27 treatment. PSL in the JCT was 2.3-fold larger in washout eyes (77.4 ± 3.3%) and 2.2-fold larger in Y27 treated eyes (75.2 ± 5.3%) versus controls (33.5 ± 5.3%, p = 0.001). Significant positive correlations were found between outflow facility and PEFL, facility and PSL and between PEFL and PSL. Our data demonstrated that similar hydrodynamic and morphological changes occurred in the aqueous humor outflow pathway of monkey eyes after induction of washout and Y27 treatment. Both Y27 and washout increase outflow facility by redistributing aqueous outflow through a larger area in the JCT. These hydrodynamic changes are likely driven by morphologic changes associated with a decrease in cell-cell and cell-matrix connections in the JCT.