humor percolates through the TM, the juxtacanalicular tissue (JCT), passes through the inner wall endothelium of Schlemm’s canal into the canal lumen, and subsequently drains into collector channels and the episcleral veins. The principal source of resistance in this pathway is at the JCT and inner wall of Schlemm’s canal. The inverse of this resistance is called outflow facility, or C in the Goldmann equation. The outflow facility, together with IOP, determines the flow rate of aqueous humor through the trabecular pathway. Outflow facility was shown to be reduced in primary open angle glaucoma (POAG) and ocular hypertensive patients (Larsson et al., 1995; Toris et al., 2002). Untreated ocular hypertensive patients also developed a progressive decrease in facility over a 10 year period (Linner, 1976).

2. Uveoscleral Pathway

In additional to the trabecular pathway, a fraction of the aqueous humor leaves the eye through the intercellular spaces of the iris root and ciliary muscle, and eventually empties into the scleral substance, the perivascular and perineural scleral spaces, and into the episcleral and orbital tissues. This outflow pathway is generally known as the uveoscleral or unconventional outflow. Its flow rate is designated as U in the Goldmann equation. At pressure levels greater than 7–10 mmHg, aqueous outflow through the uveoscleral pathway has a very low dependence of IOP, which is often not significantly diVerent from zero. This relative pressure independence is likely an integrated result of the complex resistance and capacitance characteristics of the multiple fluid compartments in the ocular tissues along the route. Recently, some feel that certain pharmacological agents, such as prostanoids, can alter uveoscleral outflow by increasing its pressure dependence (Weinreb, 2000). In ocular hypertensive patients, the calculated uveoscleral outflow rate was significantly lower than that in the age matched controls (Toris et al., 2002).

E. Measurement of Outflow Rates

Aqueous outflow in the living eye can be measured by several experimental methods. In animal studies, the most popular technique is the Ba´ra´ny’s two level constant pressure perfusion technique (Ba´ra´ny, 1964). This method requires cannulation of the anterior chamber. The inserted cannula is connected to a reservoir containing an artificial aqueous humor or other appropriate physiological solution. The reservoir is then elevated to generate raised IOP in the eye, which can be calculated by the density of the perfusion solution and the relative height of the reservoir, as well as confirmed by a second cannula connected to a pressure transducer. Driven by this pressure,

the artificial aqueous humor in the reservoir slowly flows through the outflow pathways in the eye. The flow rate can be monitored by weighing the reservoir continuously. This procedure is then repeated with a diVerent reservoir height (hence the term ‘‘two level constant pressure’’). The diVerence between the flow rates at the two pressure levels, divided by the pressure diVerence, is the outflow facility, which by definition is the pressure sensitive fraction of aqueous outflow, and mainly contributed by the trabecular outflow.

In order to directly assess uveoscleral outflow, a labeled molecule, such as 125I albumin, is injected or continuously perfused into the anterior chamber. Animals are subsequently euthanized at diVerent time points, ocular tissues of the uveoscleral pathway dissected, and levels of the labeled molecule in these tissues evaluated. Based on the initial concentration of the label in the anterior chamber and its rate of appearance in the ocular tissues, the uveoscleral outflow rate can be extrapolated (Bill, 1966). This method is cumbersome and labor intensive. It also requires careful separation of the anatomical structures involved in the two outflow pathways. An alternative method to estimate uveoscleral outflow is by subtracting the pressure dependent outflow rate from the aqueous formation rate, obtained from techniques such as the time dependent dilution of a tracer molecule in the anterior chamber. This calculated and thus indirect approximation of uveoscleral outflow is typically less accurate or reproducible than data using direct determinations.

In human subjects, a noninvasive method, based on the theoretic work of Friedenwald (Friedenwald, 1937), has been used. In this technique, a tonograph monitors the decrease in IOP continuously while a known weight rests on the cornea. From the rate of IOP change and assuming that there are no alterations in the aqueous formation rate or episcleral venous pressure, the pressure dependent aqueous outflow can be estimated. This method and its various modifications are not designed to measure uveoscleral outflow.

Based on these descriptions, it is quite clear that experimental determinations of aqueous outflow rate and facility are rather diYcult, often with questionable accuracies. Most of these techniques cannot provide a direct assessment of uveoscleral outflow. These imperfections frequently are the sources of controversies in understanding of the biochemical, cellular, and physiological mechanisms in outflow regulation, as well as the exact eVects of pharmacological agents on aqueous outflow.

F. Pathological Changes to Outflow Pathway in Glaucoma

The exact mechanism responsible for the decrease in aqueous outflow in POAG is still controversial. Nonetheless, an excessive amount of extracellular matrix (ECM) material accumulates in the TM of POAG eyes, which can

cause a partial blockade of aqueous outflow (Segawa, 1979; Rohen, 1983; Lu¨tjen Drecoll et al., 1986; Acott et al., 1988). Several biochemical changes in ECM proteins and glycosaminoglycans (GAGs) occur in the glaucomatous TM. There are increased levels of the ECM protein fibronectin (Babizhayev and Brodskaya, 1989), decreased levels of hyaluronic acid and increased chondroitin sulfate and GAG degrading enzyme resistant material (Knepper et al., 1996a), and increased levels of the ECM cross linking enzyme tissue transglutaminase (Tovar Vidales et al., 2008). In addition, aqueous humor levels of the protease inhibitor plasminogen activator inhibitor 1 (PAI 1) are elevated in glaucoma patients (Dan et al., 2005), which would also contribute to increased ECM deposition in the glaucomatous TM. Moreover, glucocorticoids, which have long been associated with POAG, also increase the synthesis and secretion of ECM molecules in cultured human and bovine TM cells and tissues (Johnson et al., 1990; Steely et al., 1992; Fujisawa, 1994; Dickerson et al., 1998). Further support for the role of the ECM in glaucoma comes from the finding that transgenic mice that have mutations introduced into the Col1A1 gene to make this collagen more resistant to degradation accumulate collagen in the aqueous outflow pathways as well as develop elevated IOP and glaucomatous optic neuropathy (Aihara et al., 2003; Mabuchi et al., 2004). In addition, inhibition of matrix metalloproteinases (MMPs), enzymes that hydrolyze ECM, in the TM elevated IOP (Bradley et al., 1998) and activation of MMPs decreased IOP (Bradley et al., 1998; Pang et al., 2003b) in perfusion cultured human eyes.

At the present time, the cause of ECM increase in the glaucomatous TM is not fully understood. Popular hypotheses include: (1) the TM cells in glaucoma patients are less active in their phagocytic activity, which leads to a reduced clearance of ECM (Bill, 1975); and (2) glaucoma patients have fewer TM cells (Alvarado et al., 1984), which can result in a slower degradation of ECM. It is also possible that other functional abnormalities of the TM cells contribute to the accumulation of ECM. Interestingly, in addition to the TM, the ciliary muscle, especially the anterior tip and the surrounding elastic fibers, of glaucoma patients has a higher amount of ECM as well (Gabelt and Kaufman, 2005).

In addition to the enhanced accumulation of ECM, abnormal cytoskeletal changes in the TM may be involved in the pathogenesis of glaucoma as well. When cultured human TM cells were exposed to glucocorticoids, the F actin microfilaments in cells progressively reorganize, forming geodesic dome like structures, called cross linked actin networks (CLANs) (Clark et al., 1994). A similar development of CLANs was also reported in outflow tissues, such as the TM and Schlemm’s canal endothelium, perfused with glucocorticoids (Clark et al., 2005). More importantly, the CLANs are more abundant in

TM cells (Clark et al., 1995) and TM tissues (Hoare et al., 2008; Read et al., 2007) derived from glaucoma donors. Furthermore, glaucoma eyes also appear to have more tangled F actin fibers in the JCT and Schlemm’s canal endothelial cells and more regions with punctuate actin distributions (Read et al., 2007). Since cytoskeleton is vital to many cell functions, these anomalies in the outflow pathways of glaucoma patients likely contribute to the reduction in outflow facility.

Genetic studies in the past decade have identified specific mutations of the myocilin gene (MYOC) that are linked directly to juvenile and adult onset POAG (Stone et al., 1997). Expression of myocilin in human TM cells can be enhanced by treatment with the glucocorticoid dexamethasone (Nguyen et al., 1998; Clark et al., 2001). Elevated IOP in MYOC glaucoma is a gain of function phenotype, since haploinsuYciency does not cause elevated IOP or glaucomatous optic neuropathy. Several recent studies indicate how MYOC mutations cause elevated IOP. Wild type (normal) myocilin is a secreted glycoprotein in the TM, and myocilin is found in the aqueous humor (Jacobson et al., 2001). Glaucomatous mutations in MYOC cause myocilin to be retained within TM cells and prevent myocilin secretion (Jacobson et al., 2001). This can lead to myocilin retention within the endoplasmic reticulum causing a stress response (Joe et al., 2003). A second study has shown that mutant myocilin interacts with PTS1R due to mutation induced misfolding and exposure of a cryptic peroxisomal targeting signal (PTS1) on the carboxy terminus of myocilin. The degree of this association between mutant myocilin and PTS1R correlates well with the clinical IOP phenotypes of MYOC glaucoma patients, with the more severe early onset POAG mutations having a higher degree of association (Shepard et al., 2007). More importantly, transduction of mouse eyes with mutant, but not wild type, human myocilin elevated IOP in mouse eyes, and IOP elevation was dependent on mutation induced exposure of the normally cryptic PTS1 signal (Shepard et al., 2007). This work provides the first true animal model of human glaucoma.

G. Current Glaucoma Therapies

Even though glaucoma manifests as an optic neuropathy and retinopathy, there are no clinically approved methods for direct neuroprotection or treatment of these aspects of the disease. Instead, all glaucoma therapies, both pharmacological and surgical, are presently directed at lowering IOP. As indicated by the abovementioned clinical trials, IOP reduction is eVective in preventing and delaying the onset and progression of glaucoma.

Pharmacological agents have been successfully used to lower pressure in the eye for more than a century. Currently, glaucoma medications can be divided into six pharmacological classes (Table I). These drugs decrease IOP by either suppressing aqueous humor production or increasing aqueous outflow (Fig. 1).

H. Aqueous Production Suppressing Agents

Compounds that are known to reduce aqueous production include the b adrenergic receptor antagonists (b blockers), carbonic anhydrase inhibitors (CAIs), and a2 adrenergic receptor agonists. They are eYcacious and safe, hence widely used in the treatment of glaucoma.

TABLE I

Current Glaucoma Therapies