Aqueous humor flows out of the eye primarily through the conventional outflow pathway that includes the trabecular meshwork and Schlemm's canal. However, a fraction of aqueous humor passes through an ...alternative or ‘unconventional’ route that includes the ciliary muscle, supraciliary and suprachoroidal spaces. From there, unconventional outflow may drain through two pathways: a uveoscleral pathway where aqueous drains across the sclera to be resorbed by orbital vessels, and a uveovortex pathway where aqueous humor enters the choroid to drain through the vortex veins. We review the anatomy, physiology and pharmacology of these pathways. We also discuss methods to determine unconventional outflow rate, including direct techniques that use radioactive or fluorescent tracers recovered from tissues in the unconventional pathway and indirect methods that estimate unconventional outflow based on total outflow over a range of pressures. Indirect methods are subject to a number of assumptions and generally give poor agreement with tracer measurements. We review the variety of animal models that have been used to study conventional and unconventional outflow. The mouse appears to be a promising model because it captures several aspects of conventional and unconventional outflow dynamics common to humans, although questions remain regarding the magnitude of unconventional outflow in mice. Finally, we review future directions. There is a clear need to develop improved methods for measuring unconventional outflow in both animals and humans.
•Aqueous humor drains through an unconventional outflow pathway that has both a uveoscleral and uveovortex component.•Tracer-based methods are required to reliably measure outflow through these pathways.•Indirect estimates of unconventional outflow generally give poor agreement with tracer-based methods.•The mouse is a promising model for studying aqueous humor outflow.•There is a need for improved measurements of unconventional outflow in both animals and humans.
The trabecular meshwork (TM) and Schlemm's canal generate the majority of outflow resistance; however, the distal regions of the conventional outflow pathway account for 25-50% of total resistance. ...Sections of distal vessels are surrounded by α-smooth muscle actin-containing cells, indicating that they may be vasoregulated. This study examined the effect of a potent vasodilator, nitric oxide (NO), and its physiological antagonist, endothelin-1 (ET-1), on the regulation of outflow resistance in the distal regions of the conventional outflow pathway. Using a physiological model of the conventional outflow pathway, human and porcine anterior segments were perfused in organ culture under constant flow conditions, while intrachamber pressure was continually monitored. For porcine anterior segments, a stable baseline outflow facility with TM intact was first achieved before anterior segments were removed and a trabeculotomy was performed. For human anterior segments, a trabeculotomy was immediately performed. In human anterior segments, 100 nM ET-1 significantly decreased distal outflow facility from 0.49 ± 0.26 to 0.31 ± 0.18 (mean ± SD) µl·min
·mmHg, P < 0.01. Perfusion with 100 µM diethylenetriamine-NO in the presence of 1 nM ET-1 immediately reversed ET-1 effects, significantly increasing distal outflow facility to 0.54 ± 0.35 µl·min
·mmHg, P = 0.01. Similar results were obtained in porcine anterior segment experiments. Therefore, data show a dynamic range of resistance generation by distal vessels in both the human and the porcine conventional outflow pathways. Interestingly, maximal contraction of vessels in the distal outflow tract of trabeculotomized eyes generated resistance very near physiological levels for both species having an intact TM.
Elevated intraocular pressure (IOP) is the predominant risk factor for glaucoma, and reducing IOP is the only successful strategy to prevent further glaucomatous vision loss. IOP is determined by the ...balance between the rates of aqueous humour secretion and outflow, and a pathological reduction in the hydraulic conductance of outflow, known as outflow facility, is responsible for IOP elevation in glaucoma. Mouse models are often used to investigate the mechanisms controlling outflow facility, but the diminutive size of the mouse eye makes measurement of outflow technically challenging. In this study, we present a new approach to measure and analyse outflow facility using iPerfusion™, which incorporates an actuated pressure reservoir, thermal flow sensor, differential pressure measurement and an automated computerised interface. In enucleated eyes from C57BL/6J mice, the flow-pressure relationship is highly non-linear and is well represented by an empirical power law model that describes the pressure dependence of outflow facility. At zero pressure, the measured flow is indistinguishable from zero, confirming the absence of any significant pressure independent flow in enucleated eyes. Comparison with the commonly used 2-parameter linear outflow model reveals that inappropriate application of a linear fit to a non-linear flow-pressure relationship introduces considerable errors in the estimation of outflow facility and leads to the false impression of pressure-independent outflow. Data from a population of enucleated eyes from C57BL/6J mice show that outflow facility is best described by a lognormal distribution, with 6-fold variability between individuals, but with relatively tight correlation of facility between fellow eyes. iPerfusion represents a platform technology to accurately and robustly characterise the flow-pressure relationship in enucleated mouse eyes for the purpose of glaucoma research and with minor modifications, may be applied in vivo to mice, as well as to eyes from other species or different biofluidic systems.
Ocular hypertension in glaucoma develops due to age-related cellular dysfunction in the conventional outflow tract, resulting in increased resistance to aqueous humor outflow. Two cell types, ...trabecular meshwork (TM) and Schlemm's canal (SC) endothelia, interact in the juxtacanalicular tissue (JCT) region of the conventional outflow tract to regulate outflow resistance. Unlike endothelial cells lining the systemic vasculature, endothelial cells lining the inner wall of SC support a transcellular pressure gradient in the basal to apical direction, thus acting to push the cells off their basal lamina. The resulting biomechanical strain in SC cells is quite large and is likely to be an important determinant of endothelial barrier function, outflow resistance and intraocular pressure. This review summarizes recent work demonstrating how biomechanical properties of SC cells impact glaucoma. SC cells are highly contractile, and such contraction greatly increases cell stiffness. Elevated cell stiffness in glaucoma may reduce the strain experienced by SC cells, decrease the propensity of SC cells to form pores, and thus impair the egress of aqueous humor from the eye. Furthermore, SC cells are sensitive to the stiffness of their local mechanical microenvironment, altering their own cell stiffness and modulating gene expression in response. Significantly, glaucomatous SC cells appear to be hyper-responsive to substrate stiffness. Thus, evidence suggests that targeting the material properties of SC cells will have therapeutic benefits for lowering intraocular pressure in glaucoma.
Aqueous humor outflow resistance is the primary determinant of intraocular pressure (IOP), and increased outflow resistance is the basis for elevated IOP associated with glaucoma. Experimental ...evidence suggests that the bulk of outflow resistance is generated in the vicinity of the inner wall endothelium of Schlemm's canal, its basement membrane and the juxtacanalicular connective tissue (JCT). However, attempts to sort out the contribution of each of these tissues to total outflow resistance have not been successful. Conventional understanding of outflow resistance assumes that the resistance of each tissue strata (i.e., the inner wall endothelium, its basement membrane and JCT) in the outflow pathway adds in series to contribute to total outflow resistance generation. However, this perspective leads to a paradox where the apparent resistances of all tissues in the outflow pathway are much lower than the measured total resistance. To resolve this paradox, we explore synergistic models of outflow resistance generation where hydrodynamic interactions between different tissue strata lead to a total resistance that is greater than the sum of the individual tissue resistances. We closely examine the “funneling” hypothesis that has emerged as a leading synergistic model, and we review the basis of funneling, mechanical and biological requirements for funneling and evidence in support of this hypothesis. We also propose refinements to the funneling model and describe how funneling may relate to segmental variability of aqueous humor outflow patterns observed within the trabecular meshwork. Pressure gradients across the JCT and inner wall endothelium will generate mechanical loads that influence the morphology of these tissues. Because tissue morphology may in turn affect outflow resistance, there exists the potential for a two-way coupling or a “fluid-solid interaction” between outflow hydrodynamics and the mechanical behavior of the inner wall and JCT. Furthermore, the adhesions and tethers between the inner wall and JCT must be physically capable of supporting such loads. We examine the structure and mechanical strength of these adhesions, and provide evidence that these adhesions and tethers are unable to support the full load imposed by the bulk of outflow resistance generation unless a substantial fraction of outflow resistance is generated within the JCT, consistent with the funneling model. This indicates that these attachments between the inner wall and JCT have considerable physiological importance for outflow resistance regulation, by maintaining the proximity between these two tissues to facilitate funneling. Further study is greatly needed to better characterize these important interactions.
Visual impairment due to glaucoma currently impacts 70 million people worldwide. While disease progression can be slowed or stopped with effective lowering of intraocular pressure, current medical ...treatments are often inadequate. Fortunately, three new classes of therapeutics that target the diseased conventional outflow tissue responsible for ocular hypertension are in the final stages of human testing. The rho kinase inhibitors have proven particularly efficacious and additive to current therapies. Unfortunately, non-contact technology that monitors the health of outflow tissue and its response to conventional outflow therapy is not available clinically. Using optical coherence tomographic (OCT) imaging and novel segmentation software, we present the first demonstration of drug effects on conventional outflow tissues in living eyes. Topical netarsudil (formerly AR-13324), a rho kinase/ norepinephrine transporter inhibitor, affected both proximal (trabecular meshwork and Schlemm’s Canal) and distal portions (intrascleral vessels) of the mouse conventional outflow tract. Hence, increased perfusion of outflow tissues was reliably resolved by OCT as widening of the trabecular meshwork and significant increases in cross-sectional area of Schlemm’s canal following netarsudil treatment. These changes occurred in conjunction with increased outflow facility, increased speckle variance intensity of outflow vessels, increased tracer deposition in conventional outflow tissues and decreased intraocular pressure. This is the first report using live imaging to show real-time drug effects on conventional outflow tissues and specifically the mechanism of action of netarsudil in mouse eyes. Advancements here pave the way for development of a clinic-friendly OCT platform for monitoring glaucoma therapy.
Nitric oxide (NO) is able to lower intraocular pressure (IOP); however, its therapeutic effects on outflow physiology are location‐ and dose‐dependent. A NO delivery platform that directly targets ...the resistance‐generating region of the conventional outflow pathway and locally liberates a controlled dose of NO is reported. An increase in outflow facility (decrease in IOP) is demonstrated in a mouse model.
Increased flow resistance is responsible for the elevated intraocular pressure characteristic of glaucoma, but the cause of this resistance increase is not known. We tested the hypothesis that ...altered biomechanical behavior of Schlemm’s canal (SC) cells contributes to this dysfunction. We used atomic force microscopy, optical magnetic twisting cytometry, and a unique cell perfusion apparatus to examine cultured endothelial cells isolated from the inner wall of SC of healthy and glaucomatous human eyes. Here we establish the existence of a reduced tendency for pore formation in the glaucomatous SC cell—likely accounting for increased outflow resistance—that positively correlates with elevated subcortical cell stiffness, along with an enhanced sensitivity to the mechanical microenvironment including altered expression of several key genes, particularly connective tissue growth factor. Rather than being seen as a simple mechanical barrier to filtration, the endothelium of SC is seen instead as a dynamic material whose response to mechanical strain leads to pore formation and thereby modulates the resistance to aqueous humor outflow. In the glaucomatous eye, this process becomes impaired. Together, these observations support the idea of SC cell stiffness—and its biomechanical effects on pore formation—as a therapeutic target in glaucoma.
Significance Glaucoma is a leading cause of blindness. The elevated intraocular pressure characteristic of many cases of glaucoma is attributable to increased resistance to aqueous humor outflow. However, the cause of this increased flow resistance has eluded investigators for over 140 y. Here we demonstrate that cells from the canal of Schlemm of glaucomatous eyes have altered gene expression and increased cytoskeletal stiffness that leads to reduced pore formation in these cells, likely accounting for increased outflow resistance associated with glaucoma. These findings thus establish that dysfunctional cytoskeletal mechanics may lie at the heart of this disease process and thereby motivate development of glaucoma therapeutics that target cell stiffness.
Catalyzed by endothelial nitric oxide (NO) synthase (eNOS) activity, NO is a gaseous signaling molecule maintaining endothelial and cardiovascular homeostasis. Principally, NO regulates the ...contractility of vascular smooth muscle cells and permeability of endothelial cells in response to either biochemical or biomechanical cues. In the conventional outflow pathway of the eye, the smooth muscle-like trabecular meshwork (TM) cells and Schlemm's canal (SC) endothelium control aqueous humor outflow resistance, and therefore intraocular pressure (IOP). The mechanisms by which outflow resistance is regulated are complicated, but NO appears to be a key player as enhancement or inhibition of NO signaling dramatically affects outflow function; and polymorphisms in NOS3, the gene that encodes eNOS modifies the relation between various environmental exposures and glaucoma. Based upon a comprehensive review of past foundational studies, we present a model whereby NO controls a feedback signaling loop in the conventional outflow pathway that is sensitive to changes in IOP and its oscillations. Thus, upon IOP elevation, the outflow pathway tissues distend, and the SC lumen narrows resulting in increased SC endothelial shear stress and stretch. In response, SC cells upregulate the production of NO, relaxing neighboring TM cells and increasing permeability of SC's inner wall. These IOP-dependent changes in the outflow pathway tissues reduce the resistance to aqueous humor drainage and lower IOP, which, in turn, diminishes the biomechanical signaling on SC. Similar to cardiovascular pathogenesis, dysregulation of the eNOS/NO system leads to dysfunctional outflow regulation and ocular hypertension, eventually resulting in primary open-angle glaucoma.
Glucocorticoid (GC) therapy is widely used to treat a variety of inflammatory diseases and conditions. While unmatched in their anti-inflammatory and immunosuppressive activities, GC therapy is often ...associated with the significant ocular side effect of GC-induced ocular hypertension (OHT) and iatrogenic open-angle glaucoma. Investigators have generated GC-induced OHT and glaucoma in at least 8 different species besides man. These models mimic many features of this condition in man and provide morphologic and molecular insights into the pathogenesis of GC-OHT. In addition, there are many clinical, morphological, and molecular similarities between GC-induced glaucoma and primary open-angle glaucoma (POAG), making animals models of GC-induced OHT and glaucoma attractive models in which to study specific aspects of POAG.
•Clinical glucocorticoid therapy can cause ocular hypertension and iatrogenic open-angle glaucoma.•Glucocorticoid-induced ocular hypertension occurs in 8 other species, from mice to nonhuman primates.•Animal models of glucocorticoid-induced ocular hypertension and glaucoma are being used to better understand this important side effect of glucocorticoid therapy.
PDF
