Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.

Decreasing outflow facility during acute IOP elevation coincides with a reduction in available area for aqueous humor outflow and the confinement of outflow to the vicinity of CC ostia. These hydrodynamic changes are likely driven by morphologic changes associated with AP collapse and herniation of IW of AP into CC ostia.

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 µM Y-27 in 7 eyes, while 5 eyes received DPBS alone. Eyes were then perfused with DPBS containing fluorescent microspheres (0.5 µm; 0.002% v/v) at a fixed volume to deliver equivalent amounts of tracer to label the hydrodynamic filtration patterns. All eyes were perfusionfixed 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±0.54 µL/min/mmHg (58.2±18.9%) while control eyes changed from 1.67±0.41 to 1.71±0.39 µl/min/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 Reprint Requests (Corresponding Author): Dr. Haiyan Gong, Department of Ophthalmology, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, Email: hgong@bu.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of th...

The session centered around three questions: What is the evidence that the pathophysiology of ocular hypertension is cell mediated, how do outflow cells deal with stress, and how does the aqueous humor enter Schlemm's canal? The discussion revealed several areas in which research could aid in our

This review traces the evolution of the concept of the blood-aqueous barrier (BAB) during the past 20 years. The classical model simply stipulated that the tight junctions of the iris vasculature and ciliary epithelium excluded plasma proteins from the aqueous humor (AH). It failed to reconcile the presence of AH protein levels equal to 1% of that found in plasma. Moreover, models of barrier kinetics assumed that the processes of AH secretion and plasma protein entry were directly linked. Thus, elevations of AH protein levels could only be explained by a pathological breakdown of the BAB. Over the last 20 years it has been shown that the plasma proteins in normal AH by-pass the posterior chamber entirely. Instead, these proteins diffuse from the capillaries of ciliary body stroma, into the iris stroma and then into the anterior chamber. This creates a reservoir of plasma-proteins in the iris stroma that is not derived from the iris vessels. This reservoir is prevented from diffusing posteriorly by tight junctions in the posterior iris epithelium. The one-way valve created by the pupil resting on the anterior lens capsule, combined with the continuous, forward flow of AH through the pupil, prevents protein reflux into the posterior chamber. Importantly, in the new paradigm, secretion of AH and the entry of plasma proteins into AH, are semi-independent events. This opens the possibility that AH protein levels could increase in the absence of breakdown of the BAB. Clinical consequences of this new paradigm of the BAB are discussed.