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Purpose: Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions)., Methods: A mathematical model of aqueous humor dynamics, including shear-mediated NO signaling, was formulated and analyzed for stability. The model includes Goldmann's equation, accounting for proximal and distal outflow resistance, and describes how elevated IOP causes narrowing of SC lumen that increases the shear stress on SC cells. Elevated shear stress stimulates NO production, which acts to reduce outflow resistance and relax trabecular meshwork cells to decrease trabecular meshwork stiffness, affecting the SC luminal caliber., Results: During constant flow perfusion, the outflow system is typically stable, returning to baseline IOP after a perturbation. In contrast, during constant pressure perfusion, the outflow system can become unstable and exhibit a time-dependent change in outflow resistance that diverges from baseline., Conclusions: The stability of shear mediated IOP homeostasis is predicted to differ critically between constant flow vs. constant pressure perfusion. Because outflow facility is typically measured at a constant pressure in the laboratory, this instability may contribute to the characteristic time-dependent increase in outflow facility, known as washout, observed in many nonhuman species. Studies of IOP homeostasis should consider how the outflow system may respond differently under constant pressure vs. constant flow perfusion.

Purpose: Intravitreal injection of drugs is commonly used for treatment of chorioretinal ocular pathologies, such as age-related macular degeneration. Injection causes a transient increase in the intraocular volume and, consequently, of the intraocular pressure (IOP). The aim of this work is to investigate how intravitreal flow patterns generated during the post-injection eye deflation influence the transport and distribution of the injected drug., Methods: We present mathematical and computational models of fluid motion and mass transport in the vitreous chamber during the transient phase after injection, including the previously unexplored effects of globe deflation as ocular volume decreases., Results: During eye globe deflation, significant fluid velocities are generated within the vitreous chamber, which can possibly contribute to drug transport. Pressure variations within the eye globe are small compared to IOP., Conclusions: Even if significant fluid velocities are generated in the vitreous chamber after drug injection, these are found to have negligible overall effect on drug distribution.

Purpose: Ocular all-trans retinoic acid (atRA) levels are influenced by visual cues, and exogenous atRA has been shown to increase eye size in chickens and guinea pigs. However, it is not clear whether atRA induces myopic axial elongation via scleral changes. Here, we test the hypothesis that exogenous atRA will induce myopia and alter scleral biomechanics in the mouse., Methods: Male C57BL/6J mice were trained to voluntarily ingest atRA + vehicle (1% atRA in sugar, 25 mg/kg) (RA: n = 16 animals) or vehicle only (Ctrl: n = 14 animals). Refractive error (RE) and ocular biometry were measured at baseline and after 1 and 2 weeks of daily atRA treatment. Eyes were used in ex vivo assays to measure scleral biomechanics (unconfined compression: n = 18), total scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 23), and specific sGAGs (immunohistochemistry: n = 18)., Results: Exogenous atRA caused myopic RE and larger vitreous chamber depth (VCD) to develop by 1 week (RE: -3.7 ± 2.2 diopters [D], P < 0.001; VCD: +20.7 ± 15.1 µm, P < 0.001), becoming more severe by 2 weeks (RE: -5.7 ± 2.2 D, P < 0.001; VCD: +32.3 ± 25.8 µm, P < 0.001). The anterior eye biometry was unaffected. While scleral sGAG content was not measurably affected, scleral biomechanics were significantly altered (tensile stiffness: -30% ± 19.5%, P < 0.001; permeability: +60% ± 95.3%, P < 0.001)., Conclusions: In mice, atRA treatment results in an axial myopia phenotype. Eyes developed myopic RE and larger VCD without the anterior eye being affected. The decrease in stiffness and increase in permeability of the sclera are consistent with the form-deprivation myopia phenotype.

Purpose: The sclera is believed to biomechanically influence eye size, facilitating the excessive axial elongation that occurs during myopigenesis. Here, we test the hypothesis that the sclera will be remodeled and exhibit altered biomechanics in the mouse model of form-deprivation (FD) myopia, accompanied by altered retinoid concentrations, a potential signaling molecule involved in the process., Methods: Male C57 Bl/6J mice were subjected to unilateral FD (n = 44 eyes), leaving the contralateral eye untreated (contra; n = 44). Refractive error and ocular biometry were measured in vivo prior to and after 1 or 3 weeks of FD. Ex vivo measurements were made of scleral biomechanical properties (unconfined compression: n = 24), scleral sulfated glycosaminoglycan (sGAG) content (dimethylmethylene blue: n = 18, and immunohistochemistry: n = 22), and ocular all-trans retinoic acid (atRA) concentrations (retina and RPE + choroid + sclera, n = 24). Age-matched naïve controls were included for some outcomes (n = 32 eyes)., Results: Significant myopia developed after 1 (-2.4 ± 1.1 diopters [D], P < 0.001) and 3 weeks of FD (-4.1 ± 0.7 D, P = 0.025; mean ± standard deviation). Scleral tensile stiffness and permeability were significantly altered during myopigenesis (stiffness = -31.4 ± 12.7%, P < 0.001, and permeability = 224.4 ± 205.5%, P < 0.001). Total scleral sGAG content was not measurably altered; however, immunohistochemistry indicated a sustained decrease in chondroitin-4-sulfate and a slower decline in dermatan sulfate. The atRA increased in the retinas of eyes form-deprived for 1 week., Conclusions: We report that biomechanics and GAG content of the mouse sclera are altered during myopigenesis. All scleral outcomes generally follow the trends found in other species and support a retina-to-sclera signaling cascade underlying mouse myopigenesis.

Purpose: Nonarteritic anterior ischemic optic neuropathy (NAION) has been associated with a thickened choroid at the optic nerve head (ONH). Here, we use computational modeling to better understand how choroidal expansion and choroidal geometry influence tissue deformation within the ONH relative to intraocular pressure (IOP) and intracranial pressure (ICP) effects., Methods: Using a model of the posterior eye that included the sclera, peripapillary sclera, annular ring, pia mater, dura mater, neural tissues, Bruch's membrane, choroid, and lamina cribrosa, we examined how varying material properties of ocular tissues influenced ONH deformations under physiological and supra-physiological, or "pathological," conditions. We considered choroidal expansion (c. 35 µL of expansion), elevated IOP (30 mm Hg), and elevated ICP (20 mm Hg), and calculated peak strains in the ONH relative to a baseline condition representing an individual in the upright position., Results: Supra-physiological choroidal expansion had the largest impact on strains in the prelaminar neural tissue. In addition, compared to a tapered choroid, a "blunt" choroid insertion at the ONH resulted in higher strains. Elevated IOP and ICP caused the highest strains within the lamina cribrosa and retrolaminar neural tissue, respectively., Conclusions: Acute choroidal expansion caused large deformations of the ONH and these deformations were impacted by choroid geometry. These results are consistent with the concept that compartment syndrome due to the choroid geometry and/or expansion at the ONH contributes to NAION. Prolonged deformations due to supra-physiological loading may induce a mechanobiological response or ischemia, highlighting the potential impact of choroidal expansion on biomechanical strains in the ONH.

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.

Purpose: Rodent and primate models are commonly used in glaucoma research; however, both have their limitations. The tree shrew (Tupaia belangeri) is an emerging animal model for glaucoma research owing in part to having a human-like optic nerve head anatomy, specifically a collagenous load-bearing lamina. However, the anterior segment anatomy and function have not been extensively studied in the tree shrew. Thus, the purpose of this study was to provide the first detailed examination of the anterior segment anatomy and aqueous outflow facility in the tree shrew., Methods: Aqueous outflow dynamics were measured in five ostensibly normal eyes from three tree shrews using the iPerfusion system over a range of pressures. Gross histological assessment and immunohistochemistry were performed to characterize anterior segment anatomy and to localize several key molecules related to aqueous outflow., Results: Anterior segment anatomy in tree shrews is similar to humans, demonstrating a scleral spur, a multilayered trabecular meshwork and a circular Schlemm's canal with a single lumen. Average outflow facility was 0.193 µL/min/mm Hg (95% confidence interval, 0.153-0.244), and was stable over time. Outflow facility was more similar between contralateral eyes (approximately 5% average difference) than between eyes of different animals. No significant dependence of outflow facility on time or pressure was detected (pressure-flow nonlinearity parameter of 0.01 (95% % confidence interval, -0.29 to 0.31 CI µL/min/mm Hg)., Conclusions: These studies lend support to the usefulness of the tree shrew as a novel animal model in anterior segment glaucoma and pharmacology research. The tree shrew's cost, load-bearing collagenous lamina cribrosa, and lack of washout or anterior chamber deepening provides a distinct experimental and anatomic advantage over the current rodent and nonhuman primate models used for translational research.

Purpose: Re-cellularization of the trabecular meshwork (TM) using stem cells is a potential novel treatment for ocular hypertension associated with glaucoma. To assess the therapeutic efficacy of this approach, improved in vivo and ex vivo models of TM pathophysiology are needed. Here, we investigate whether oxidative stress, induced by hydrogen peroxide (H2O2), can model glaucomatous ocular hypertension in the readily available porcine anterior segment organ culture model., Methods: The impact of H2O2 on TM cell viability and function was first evaluated in vitro using primary porcine TM cells. Oxidative stress was then induced by H2O2 infusion into perfused porcine anterior segments. Trabecular meshwork function was assessed by tracking matrix metalloproteinase (MMP) activity and the ability of the preparation to maintain intraocular pressure (IOP) homeostasis after a flow challenge (doubled fluid infusion rate). Finally, the TM was evaluated histologically., Results: H2O2 treatment resulted in a titratable reduction in cellularity across multiple primary TM cell donor strains. In organ culture preparations, H2O2-treated eyes showed impaired IOP homeostasis (i.e., IOPs stabilized at higher levels after a flow challenge vs. control eyes). This result was consistent with reduced MMP activity and TM cellularity; however, damage to the TM microstructure was not histologically evident in anterior segments receiving H2O2., Conclusions: Titrated H2O2 infusion resulted in TM cellular dysfunction without destruction of TM structure. Thus, this porcine organ culture model offers a useful platform for assessing trabecular meshwork therapies to treat ocular hypertension associated with glaucoma.

Purpose: Glaucoma is the second leading cause of blindness worldwide. Recent work suggests that estrogen and the timing of menopause play a role in modulating the risk of developing glaucoma. Menopause is known to cause modest changes in intraocular pressure; yet, whether this change is mediated through the outflow pathway remains unknown. Menopause also affects tissue biomechanical properties throughout the body; however, the impact of menopause on ocular biomechanical properties is not well characterized., Methods: Here, we simultaneously assessed the impact of menopause on aqueous outflow facility and ocular compliance, as a measure of corneoscleral shell biomechanics. We used young (3-4 months old) and middle-aged (9-10 months old) Brown Norway rats. Menopause was induced by ovariectomy (OVX), and control animals underwent sham surgery, resulting in the following groups: young sham (n = 5), young OVX (n = 6), middle-aged sham (n = 5), and middle-aged OVX (n = 5). Eight weeks postoperatively, we measured outflow facility and ocular compliance., Results: Menopause resulted in a 34% decrease in outflow facility and a 19% increase in ocular compliance (P = 0.011) in OVX animals compared with sham controls (P = 0.019)., Conclusions: These observations reveal that menopause affects several key physiological factors known to be associated with glaucoma, suggesting that menopause may contribute to an increased risk of glaucoma in women.

Purpose: Choroid geometry and swelling have been proposed to contribute to ocular pathologies. Thus, it is important to understand how the choroid may impact the optic nerve head (ONH) biomechanical environment. We developed a finite element model to study how acute choroidal swelling and choroid geometry affect ONH deformation., Methods: We developed two geometric models of the ONH: one with a "blunt" choroidal insertion and another with a "tapered" choroid insertion. We examined how choroidal volume changes (2.1-14.2 μL, estimated to occur during the ocular pulse) impact biomechanical strain in three tissue regions: the prelaminar neural tissue, lamina cribrosa, and retrolaminar neural tissue. Then, we performed a sensitivity analysis to understand how variation in ONH pressures, tissue material properties, and choroidal swelling influenced the peak tissue strains., Results: Choroidal swelling in the blunt choroid geometry had a large impact on the strains in the prelaminar neural tissue, with magnitudes comparable to those expected to occur due to an IOP of 30 mm Hg. Choroidal swelling in the tapered choroid geometry also affected strains but to a lesser extent compared to the blunt geometry. A sensitivity analysis confirmed that choroidal swelling was more influential on prelaminar neural tissue strains in the blunt choroid geometry., Conclusions: Choroid anatomy and swelling can interact to play an important role in prelaminar neural tissue deformation. These findings suggest that the choroid may play an important, and previously unappreciated, role in ONH biomechanics, and motivate additional research to better define the in vivo effects of choroidal volume change.

Purpose: The purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements., Methods: Postmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated. TM stiffness was deduced by an inverse finite element modeling (FEM) approach. A series of AFM forcemaps was acquired along a line traversing the anterior angle on a radially cut flat-mount corneoscleral wedge with TM facing upward., Results: The elastic modulus of normal hTM estimated by inverse FEM was 70 ± 20 kPa (mean ± SD), whereas glaucomatous hTM was slightly stiffer (98 ± 19 kPa). This trend was consistent with TM stiffnesses measured by AFM: normal hTM stiffness = 1.37 ± 0.56 kPa, which was lower than glaucomatous hTM stiffness (2.75 ± 1.19 kPa). None of these differences were statistically significant. TM in HF wedges was softer than that in LF wedges for both normal and glaucomatous eyes based on the inverse FEM approach but not by AFM. Outflow facility was significantly correlated with TM stiffness estimated by FEM in six human eyes (P = 0.018)., Conclusions: TM stiffness is higher, but only modestly so, in glaucomatous patients. Outflow facility in both normal and glaucomatous human eyes appears to associate with TM stiffness. This evidence motivates further studies to investigate factors underlying TM biomechanical property regulation.

Purpose: Cerebrospinal fluid pressure (CSFp) changes are involved or implicated in various ocular conditions including glaucoma, idiopathic intracranial hypertension, and visual impairment and intracranial pressure syndrome. However, little is known about the effects of CSFp on lamina cribrosa and retrolaminar neural tissue (RLNT) biomechanics, potentially important in these conditions. Our goal was to use an experimental approach to visualize and quantify the deformation of these tissues as CSFp increased., Methods: The posterior eye and RLNT of porcine eyes (n = 3) were imaged using synchrotron radiation phase-contrast micro-computed tomography (PC μCT) at an intraocular pressure of 15 mm Hg and CSFps of 4, 10, 20, and 30 mm Hg. Scans of each tissue region were acquired at each CSFp step and analyzed using digital volume correlation to determine 3-dimensional tissue deformations., Results: Elevating CSFp increased the strain in the lamina cribrosa and RLNT of all three specimens, with the largest strains occurring in the RLNT. Relative to the baseline CSFp of 4 mm Hg, at 30 mm Hg, the lamina cribrosa experienced a mean first and third principal strain of 4.4% and -3.5%, respectively. The corresponding values for the RLNT were 9.5% and -9.1%., Conclusions: CSFp has a significant impact on the strain distributions within the lamina cribrosa and, more prominently, within the RLNT. Elevations in CSFp were positively correlated with increasing deformations in each region and may play a role in ocular pathologies linked to changes in CSFp.

Purpose: To determine clearance kinetics and routes of clearance of molecules from the suprachoroidal space (SCS) of live New Zealand White rabbits., Methods: Suprachoroidal space collapse rate and pressure changes after microneedle injection into SCS were determined. Fluorescent fundus images were acquired to determine clearance rates of molecules ranging in size from 332 Da to 2 MDa. Microneedle injections of fluorescein were performed, and samples were taken from various sites over time to determine amount of fluorescein exiting the eye. Clearance transport was modeled theoretically and compared with experimental data., Results: After injection, pressures in SCS and vitreous humor spiked and returned to baseline within 20 minutes; there was no difference between these two pressures. Suprachoroidal space collapse occurred within 40 minutes. One hour after fluorescein injection, 46% of fluorescein was still present in the eye, 15% had transported across sclera, 6% had been cleared by choroidal vasculature, and 4% had exited via leakage pathways. Characteristic clearance time increased in proportion with molecular radius, but total clearance of 2 MDa FITC-dextran was significantly slower (21 days) than smaller molecules. These data generally agreed with predictions from a theoretical model of molecular transport., Conclusions: Guided by experimental data in the context of model predictions, molecular clearance from SCS occurred in three regimes: (1) on a time scale of approximately 10 minutes, fluid and molecules exited SCS by diffusion into sclera and choroid, and by pressure-driven reflux via transscleral leakage sites; (2) in approximately 1 hour, molecules cleared from choroid by blood flow; and (3) in 1 to 10 hours, molecules cleared from sclera by diffusion and convection.

Purpose: Scleral stiffening has been proposed as a treatment for glaucoma to protect the lamina cribrosa (LC) from excessive intraocular pressure-induced deformation. Here we experimentally evaluated the effects of moderate stiffening of the peripapillary sclera on the deformation of the LC., Methods: An annular sponge, saturated with 1.25% glutaraldehyde, was applied to the external surface of the peripapillary sclera for 5 minutes to stiffen the sclera. Tissue deformation was quantified in two groups of porcine eyes, using digital image correlation (DIC) or computed tomography imaging and digital volume correlation (DVC). In group A (n = 14), eyes were subjected to inflation testing before and after scleral stiffening. Digital image correlation was used to measure scleral deformation and quantify the magnitude of scleral stiffening. In group B (n = 5), the optic nerve head region was imaged using synchrotron radiation phase-contrast microcomputed tomography (PC μCT) at an isotropic spatial resolution of 3.2 μm. Digital volume correlation was used to compute the full-field three-dimensional deformation within the LC and evaluate the effects of peripapillary scleral cross-linking on LC biomechanics., Results: On average, scleral treatment with glutaraldehyde caused a 34 ± 14% stiffening of the peripapillary sclera measured at 17 mm Hg and a 47 ± 12% decrease in the maximum tensile strain in the LC measured at 15 mm Hg. The reduction in LC strains was not due to cross-linking of the LC., Conclusions: Peripapillary scleral stiffening is effective at reducing the magnitude of biomechanical strains within the LC. Its potential and future utilization in glaucoma axonal neuroprotection requires further investigation.

Purpose: Visual impairment and intracranial pressure (VIIP) syndrome is a health concern for long-duration spaceflight, and a proposed risk factor is elevation of intracranial pressure (ICP). Our goal was to use finite element modeling to simulate how elevated ICP and interindividual differences affect tissue deformation within the optic nerve head (ONH)., Methods: We considered three ICP conditions: the upright and supine position on earth and an elevated ICP assumed to occur in chronic microgravity. Within each condition we used Latin hypercube sampling to consider a range of pressures and ONH tissue mechanical properties, determining the influence of each input on the following outcome measures: peak strains in the prelaminar tissue, lamina cribrosa, and retrolaminar optic nerve. Elevated strains can alter cell phenotype and induce tissue remodeling., Results: Elevating ICP increased the strains in the retrolaminar optic nerve. Variations in IOP, ICP, and in optic nerve and lamina cribrosa stiffness had the strongest influence on strains within the ONH. We predicted that 5% to 47% of individuals in microgravity would experience peak strains in the retrolaminar optic nerve larger than expected on earth. Having a soft optic nerve or pia mater and elevated ICP were identified as risk factors for these "extreme" strains., Conclusions: Intracranial pressure and mechanical properties of the ONH influence the risk for experiencing extreme strains in the retrolaminar optic nerve. These extreme strains may activate mechanosensitive cells that induce tissue remodeling and are a risk factor for the development of VIIP. Future studies must also consider variations in ONH anatomy.

Purpose: Mice are commonly used to study conventional outflow physiology. This study examined how physical factors (hydration, temperature, and anterior chamber [AC] deepening) influence ocular perfusion measurements in mice., Methods: Outflow facility (C) and pressure-independent outflow (Fu) were assessed by multilevel constant pressure perfusion of enucleated eyes from C57BL/6 mice. To examine the effect of hydration, seven eyes were perfused at room temperature, either immersed to the limbus in saline and covered with wet tissue paper or exposed to room air. Temperature effects were examined in 12 eyes immersed in saline at 20 °C or 35 °C. Anterior chamber deepening was examined in 10 eyes with the cannula tip placed in the anterior versus posterior chamber (PC). Posterior bowing of the iris (AC deepening) was visualized by three-dimensional histology in perfusion-fixed C57BL/6 eyes and by spectral-domain optical coherence tomography in living CD1 mice., Results: Exposure to room air did not significantly affect C, but led to a nonzero Fu that was significantly reduced upon immersion in saline. Increasing temperature from 20 °C to 35 °C increased C by 2.5-fold, more than could be explained by viscosity changes alone (1.4-fold). Perfusion via the AC, but not the PC, led to posterior iris bowing and increased outflow., Conclusions: Insufficient hydration contributes to the appearance of pressure-independent outflow in enucleated mouse eyes. Despite the large lens, AC deepening may artifactually increase outflow in mice. Temperature-dependent metabolic processes appear to influence conventional outflow regulation. Physical factors should be carefully controlled in any outflow studies involving mice.

Purpose: Previous studies have suggested that the lamina cribrosa (LC) and its surrounding sclera are biomechanically important in the pathogenesis of glaucoma, but many were limited by assumptions of tissue isotropy and homogeneity. Here, we used an improved biomechanical model driven by experimental measurements of scleral and LC collagen fiber organization to more accurately evaluate optic nerve head (ONH) biomechanics., Methods: Collagen fiber organization was quantitatively mapped across human ONH cryosections (three normal and three glaucomatous) using small-angle light scattering (SALS) and fed into two-dimensional finite element models loaded under biaxial stress to simulate raised intraocular pressure. Effects of artificial variations in collagen fiber microstructure and stiffness on LC and scleral strains were also investigated., Results: Scleral collagen fibers were circumferential and exhibited the highest alignment in a region not immediately adjacent to, but at a distance (400-500 μm) away from, the LC. In models, such a fiber arrangement yielded rings of low strain (second principal and effective) in the scleral region immediately adjacent to the LC. Further sensitivity analyses showed that scleral fiber alignment was crucial in determining LC strain levels. Moderate scleral anisotropy (as observed physiologically) was more effective than isotropy or high anisotropy in limiting LC and scleral strain magnitude., Conclusions: The presence of a heterogeneous collagen fiber organization in the peripapillary sclera appears effective in limiting LC strain and is able to reduce strain levels at the scleral canal boundary: a transition zone prone to LC disinsertion, focal lamina cribrosa defects, and optic disc hemorrhages in glaucoma., (Copyright 2015 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Endothelial nitric oxide (NO) synthase is regulated by shear stress. At elevated intraocular pressures when the Schlemm's canal (SC) begins to collapse, shear stress is comparable with that in large arteries. We investigated the relationship between NO production and shear stress in cultured human SC cells., Methods: Schlemm's canal endothelial cells isolated from three normal and two glaucomatous human donors were seeded into Ibidi flow chambers at confluence, cultured for 7 days, and subjected to steady shear stress (0.1 or 10 dynes/cm(2)) for 6, 24, or 168 hours. Cell alignment with flow direction was monitored, and NO production was measured using 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) and Griess reagents. Human trabecular meshwork (TM) and umbilical vein endothelial cells (HUVECs) were used as controls., Results: Normal SC strains aligned with the direction of flow by 7 days. Comparing 0.1 vs. 10 dynes/cm(2), NO levels increased by 82% at 24 hours and 8-fold after 7 days by DAF-FM, and similar results were obtained with Griess reagent. Shear responses by SC cells at 24 hours were comparable with HUVECs, and greater than TM cells, which appeared shear-insensitive. Nitric oxide production by SC cells was detectable as early as 6 hours and was inhibited by 100 μM nitro-L-arginine methyl ester. Two glaucomatous SC cell strains were either unresponsive or lifted from the plate in the face of shear., Conclusions: Shear stress triggers NO production in human SC cells, similar to other vascular endothelia. Increased shear stress and NO production during SC collapse at elevated intraocular pressures may in part mediate IOP homeostasis., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: To determine whether dexamethasone (DEX)-induced ocular hypertension (OHT) in mice mimics the hallmarks of steroid-induced glaucoma (SIG) in humans, including reduced conventional outflow facility (C), increased extracellular matrix (ECM), and myofibroblasts within the outflow pathway., Methods: Osmotic mini-pumps were implanted subcutaneously into C57BL/6J mice for systemic delivery of DEX (3-4 mg/kg/d, n = 31 mice) or vehicle (n = 28). IOP was measured weekly by rebound tonometry. After 3 to 4 weeks, mice were euthanized and eyes enucleated for ex vivo perfusion to measure C, for electron microscopy to examine the trabecular meshwork (TM) and Schlemm's canal (SC), or for immunohistochemistry to examine type IV collagen and α-smooth muscle actin. The length of basement membrane material (BMM) was measured along the anterior-posterior extent of SC by electron microscopy. Ultrastructural changes in BMM of DEX-treated mice were compared against archived human SIG specimens., Results: Dexamethasone increased IOP by 2.6 ± 1.6 mm Hg (mean ± SD) over 3 to 4 weeks and decreased C by 52% ± 17% versus controls. Intraocular pressure elevation correlated with decreased C. Dexamethasone treatment led to increased fibrillar material in the TM, plaque-like sheath material surrounding elastic fibers, and myofibroblasts along SC outer wall. The length of BMM underlying SC was significantly increased in mice with DEX and in humans with SIG, and in mice decreased C correlated with increased BMM., Conclusions: Dexamethasone-induced OHT in mice mimics hallmarks of human SIG within 4 weeks of DEX treatment. The correlation between reduced C and newly formed ECM motivates further study using DEX-treated mice to investigate the pathogenesis of conventional outflow obstruction in glaucoma., (Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.)

Purpose: Mouse models are useful for glaucoma research, but it is unclear whether intraocular pressure (IOP) regulation in mice operates through mechanisms similar to those in humans. Our goal was to determine whether pharmacologic compounds that affect conventional outflow facility in human eyes exert similar effects in C57BL/6 mice., Methods: A computerized perfusion system was used to measure conventional outflow facility in enucleated mouse eyes ex vivo. Paired eyes were perfused sequentially, either immediately after enucleation or after 3 hours storage at 4°C. Three groups of experiments examined sphingosine 1-phosphate (S1P), S1P with antagonists to S1P(1) and S1P(2) receptors, and the prostanoid EP(4) receptor agonist 3,7-dithia PGE(1). We also examined whether a 24-hour postmortem delay affected the response to 3,7-dithia prostaglandin E(1) (PGE(1))., Results: S1P decreased facility by 39%, and was blocked almost completely by an S1P(2), but not S1P(1), receptor antagonist. The S1P(2) receptor antagonist alone increased facility nearly 2-fold. 3,7-dithia PGE(1) increased facility by 106% within 3 hours postmortem. By 24 hours postmortem, the facility increase caused by 3,7-dithia PGE(1) was reduced 3-fold, yet remained statistically detectable., Conclusions: C57BL/6 mice showed opposing effects of S1P(2) and EP(4) receptor activation on conventional outflow facility, as observed in human eyes. Pharmacologic effects on facility were detectable up to 24 hours postmortem in enucleated mouse eyes. Mice are suitable models to examine the pharmacology of S1P and EP(4) receptor stimulation on IOP regulation as occurs within the conventional outflow pathway of human eyes, and are promising for studying other aspects of aqueous outflow dynamics.

Purpose: Previous work has suggested a major role of scleral biomechanics in the pathogenesis of glaucoma. Since fiber orientation in connective tissues is a key determinant of tissue biomechanics, experimental characterization of scleral fiber orientation is needed to fully understand scleral biomechanics. This is a report of baseline experimental measurements of fiber orientation in whole normal rat scleras., Methods: Twenty ostensibly normal Norway brown rat eyes were fixed in 4% paraformaldehyde. The scleras were cleaned of intra- and extraorbital tissues and dissected into five patches, and each patch was glycerol treated to maximize its transparency. Fiber orientation was measured using small-angle light scattering (SALS). Scattering patterns were analyzed to extract two microstructural parameters at each measurement location-the preferred fiber orientation and the degree of alignment-yielding a fiber orientation map for each sclera., Results: Rat sclera is structurally anisotropic with several consistent features. At the limbus, fibers were highly aligned and organized primarily into a distinct ring surrounding the cornea. In the equatorial region, the fibers were primarily meridionally aligned. In the posterior and peripapillary region, the scleral fibers were mostly circumferential but less aligned than those in the anterior and equatorial regions., Conclusions: Circumferential scleral fibers may act as reinforcing rings to limit corneal and optic nerve head deformations, whereas equatorial meridional fibers may either provide resistance against extraocular muscle forces or limit globe axial elongation.

Purpose: Pathology in the primary drainage pathway for aqueous humor in the eye is responsible for ocular hypertension, the only treatable risk factor in patients with glaucoma. Unfortunately, the mechanisms that regulate pressure-dependent drainage of aqueous humor and thus intraocular pressure (IOP) are unknown. To better understand one possible underlying molecular factor that regulates IOP, nitric oxide (NO), pressure-dependent drainage in transgenic mice overexpressing endothelial NO synthase (eNOS) was studied., Methods: IOP was measured by rebound tonometry in mice, and pressure versus flow data were measured by ex vivo perfusion at multiple pressures between 8 and 45 mm Hg, using mock AH ±100 μM L-NAME. A subset of eyes was examined histologically using standard techniques or was assayed for fusion protein expression by Western blot analysis., Results: IOP was lower (9.6 ± 2.7 vs. 11.4 ± 2.5 mm Hg; mean ± SD; P = 0.04) and pressure-dependent drainage was higher (0.0154 ± 0.006 vs. 0.0066 ± 0.0009 μL/min/mm Hg; P = 0.002) in the transgenic mice than in the wild-type animals; however, pressure-independent drainage was unaffected. The NOS inhibitor L-NAME normalized pressure-dependent drainage in transgenic animals. For IOP >35 mm Hg, the slope of the pressure-flow curve in wild-type mice increased to match that seen in transgenic mice. Shear stress in the pressure-dependent pathway at elevated pressures was calculated to be in a range known to affect eNOS expression and activity in vascular endothelia., Conclusions: Endothelial NOS overexpression lowers IOP by increasing pressure-dependent drainage in the mouse eye. Data are consistent with NO's having a mechanoregulatory role in aqueous humor dynamics, with eNOS induction at elevated IOPs leading to increased pressure-dependent outflow.

Purpose: To improve the quality of optical coherence tomography (OCT) images of the optic nerve head (ONH)., Methods: Two algorithms were developed, one to compensate for light attenuation and the other to enhance contrast in OCT images. The former was borrowed from developments in ultrasound imaging and was proven suitable with either time- or spectral-domain OCT. The latter was based on direct application of pixel intensity exponentiation. The performances of these two algorithms were tested on spectral-domain OCT images of four adult ONHs., Results: Application of the compensation algorithm significantly reduced the intralayer contrast (from 0.74 ± 0.16 to 0.17 ± 0.12; P < 0.001), indicating successful blood vessel shadow removal. Furthermore, compensation dramatically improved the visibility of deeper ONH tissues, such as the peripapillary sclera and lamina cribrosa. Application of the contrast-enhancement algorithm significantly increased the interlayer contrast (from 0.48 ± 0.22 to a maximum of 0.89 ± 0.05; P < 0.001) and thus allowed a better differentiation of tissue boundaries. Contrast enhancement was robust only when compensation was considered., Conclusions: The proposed algorithms are simple and can significantly improve the quality of ONH images clinically captured with OCT. This study has important implications, as it will help improve our ability to perform automated segmentation of the ONH; quantify the morphometry and biomechanics of ONH tissues in vivo; and identify potential risk indicators for glaucoma.

Purpose: The response of cells (e.g., optic nerve head [ONH] cells) to mechanical stress is important in glaucoma. Studies have reported the biological effects of hydrostatic pressure on ONH cells cultured on a rigid substrate. An apparatus, designed to independently vary hydrostatic pressure and gas tension (including oxygen tension) in culture medium, was used to evaluate the effects of pressure and tension on cell migration, shape, and α-tubulin architecture in a transformed cell line (DITNC1 rat cortical astrocytes)., Methods: During the assay period, cells were exposed to one of four experimental configurations: (1) control pressure and control gas tension; (2) high-pressure (7.4 mm Hg) and reduced gas tension; (3) control pressure and reduced gas tension; and (4) high-pressure and control gas tension., Results: Calculations suggested that the cells in configurations 2 and 3 were hypoxic, as confirmed by direct measurements in configuration 2. No effects of hydrostatic pressure were observed on cell migration or α-tubulin architecture. However, cells cultured under low gas tension (configurations 2 and 3) showed increased migration at 48 and 72 hours (P < 0.05)., Conclusions: A hydrostatic pressure of 7.4 mm Hg has no effect on DITNC1 astrocytes cultured on rigid coverslips, whereas hypoxia associated with a fluid column creating this pressure does. These results differ from those in a previous report, the results of which may be explained by altered gas tensions in the culture medium. Steps are recommended for control of secondary effects when testing the effect of pressure on cultured cells.

Purpose: Mice are commonly used in glaucoma research, but relatively little is known about aqueous outflow dynamics in the species. To facilitate future use of the mouse as a model of aqueous humor outflow, several fundamental physiological parameters were measured in the mouse eye., Methods: Eyes from adult mice of either sex (C57BL/6 background) were enucleated, cannulated with a 33-gauge needle, and perfused at constant pressure while inflow was continuously measured., Results: At 8 mm Hg, total outflow facility (C(total)) was 0.022 ± 0.005 μL/min/mm Hg (all values mean ± SD; n = 21). The flow-pressure relationship was linear up to 35 mm Hg. The conventional outflow facility (C(conv)) was 0.0066 ± 0.0009 μL/min/mm Hg, and the unconventional outflow (F(u)) was 0.114 ± 0.019 μL/min, both measured at room temperature. At 8 mm Hg, 66% of the outflow was via the unconventional pathway. In a more than 2-hour-long perfusion at 8 mm Hg, the rate of facility change was 2.4% ± 5.4% (n = 11) of starting facility per hour. The ocular compliance (0.086 ± 0.017 μL/mm Hg; n = 5) was comparable to the compliance of the perfusion system (0.100 ± 0.004 μL/mm Hg)., Conclusions: Mouse eyes are similar to human eyes, in that they have no detectable washout rate and a linear pressure-flow relationship over a broad range of intraocular pressures. Because of the absence of washout and the apparent presence of a true Schlemm's canal, the mouse is a useful model for studying the physiology of the inner wall of Schlemm's canal and the conventional outflow tissues.

Purpose: The authors sought to develop a technique for isolating and culturing angular aqueous plexus (AAP) cells from more plentiful porcine eyes. AAP is an analogue of Schlemm's canal., Methods: Cells were differentially selected with puromycin, a toxin often used to select brain microvascular endothelial cells based on the expression of P-glycoprotein (P-gp), a multidrug resistance efflux pump. Trabecular meshwork containing AAP was dissected and pooled from fresh porcine eyes, digested in collagenase I, washed, filtered, and cultured for 8 days in a gelatin-coated plastic flask. Cells were then selected by exposure to 4 μg/mL puromycin for 2 days in the culture medium. Cells were fixed and immunostained for P-gp, ICAM II, von Willebrand factor (vWF), VE-cadherin, and α-smooth muscle actin (α-SMA)., Results: Histology of the limbus showed that the dissection was limited to the trabecular meshwork region, including the AAP. Before puromycin treatment, cells appeared heterogeneous and polygonal, suggestive of a mixed population. More than 90% of the cells were removed by puromycin, leaving a population that appeared uniformly cobblestone-like when grown to confluence and that was contact inhibited. Puromycin-selected cells stained positively for the endothelial markers ICAM II, vWF, and VE-cadherin but negatively for α-SMA, consistent with staining patterns in whole tissue., Conclusions: Based on marker expression, morphology, and behavior in culture, puromycin-selected cells from porcine outflow tissues are AAP endothelial cells. Thus, porcine eyes can provide a plentiful alternative cell source for studying Schlemm's canal biology related to ocular hypertension.

Purpose: To determine the effect of the F-actin-disrupting agent latrunculin-B on aqueous outflow facility and trabecular meshwork architecture in human eyes., Methods: After baseline facility measurement in human eye bank eyes (n = 9 pairs), one eye of each pair received anterior chamber exchange and continued perfusion with medium containing 1 microM latrunculin-B. Contralateral eyes were treated in a similar manner with vehicle. Eyes were fixed by anterior chamber exchange and perfusion with universal fixative at 8 mm Hg (corresponding to a physiologic pressure of 15 mm Hg in vivo), and outflow pathway tissues were examined by transmission and scanning electron microscopy., Results: Perfusion of eyes with 1 microM latrunculin-B caused a continuous and ongoing increase in outflow facility, resulting in a net facility difference of 64% 2 hours after drug administration (P < 0.006). Transmission electron microscopy showed subtle and focal detachment of the inner wall of Schlemm's canal, rarefaction of the juxtacanalicular tissue (JCT), and cell-cell and cell-matrix detachment. Scanning electron microscopy showed collapsed vacuoles in the inner wall of Schlemm's canal and a marked increase in the number and size of border (paracellular) pores in the inner wall., Conclusions: Latrunculin-B increases outflow facility in postmortem human eyes. The mechanism of facility increase is most likely due to loss of mechanical integrity of the trabecular meshwork as a consequence of reduction in cell-cell and cell-matrix adhesion. The facility increase and the extent of inner wall separation from the JCT that we observed were both qualitatively similar to that reported in living monkey eyes, but the magnitude of the facility increase and morphologic changes were much less than in the living monkey. This supports the idea that inner wall separation from the JCT may modulate outflow facility.

Purpose: The biomechanical environment within the optic nerve head (ONH) may play a role in retinal ganglion cell loss in glaucomatous optic neuropathy. This was a systematic analysis in which finite element methods were used to determine which anatomic and biomechanical factors most influenced the biomechanical response of the ONH to acute changes in IOP., Methods: Based on a previously described computational model of the eye, each of 21 input factors, representing the biomechanical properties of relevant ocular tissues, the IOP, and 14 geometric factors were independently varied. The biomechanical response of the ONH tissues was quantified through a set of 29 outcome measures, including peak and mean stress and strain within each tissue, and measures of geometric changes in ONH tissues. Input factors were ranked according to their aggregated influence on groups of outcome measures., Results: The five input factors that had the largest influence across all outcome measures were, in ranked order: stiffness of the sclera, radius of the eye, stiffness of the lamina cribrosa, IOP, and thickness of the scleral shell. The five least influential factors were, in reverse ranked order: retinal thickness, peripapillary rim height, cup depth, cup-to-disc ratio, and pial thickness. Factor ranks were similar for various outcome measure groups and factor ranges., Conclusions: The model predicts that ONH biomechanics are strongly dependent on scleral biomechanical properties. Acute deformations of ONH tissues, and the consequent high levels of neural tissue strain, were less strongly dependent on the action of IOP directly on the internal surface of the ONH than on the indirect effects of IOP on the sclera. This suggests that interindividual variations in scleral properties could be a risk factor for the development of glaucoma. Eye size and lamina cribrosa biomechanical properties also have a strong influence on ONH biomechanics.

Purpose: Biomechanical factors have been implicated in the development of glaucomatous optic neuropathy, particularly at the level of the lamina cribrosa. The goal of this study was to characterize the biomechanics of the optic nerve head using computer modeling techniques., Methods: Several models of the optic nerve head tissues (pre- and postlaminar neural tissue, lamina cribrosa, central retinal vessel, sclera, and pia mater) were constructed. Stresses, deformations, and strains were computed using finite element modeling for a range of normal and elevated intraocular pressures. Computed retinal surface deformations were compared with measured deformation patterns in enucleated human eyes. A sensitivity analysis was performed in which tissue properties and selected geometric features were varied., Results: Acute IOP-induced deformation of the vitreoretinal interface was highly dependent on optic cup shape but showed a characteristic "W-shaped" profile that did not match the deformation of the anterior surface of the lamina cribrosa. The central retinal vasculature had surprisingly little effect on optic nerve head biomechanics. At an IOP of 50 mm Hg, strains (fractional elongation) in the lamina cribrosa averaged 4% to 5.5%, dependent on model geometry, with maximum strains up to 7.7%. Strains in the lamina cribrosa were more dependent on scleral stiffness, scleral thickness, and scleral canal diameter than on lamina cribrosa stiffness and optic cup shape., Conclusions: Computed levels of strain in the lamina cribrosa are biologically significant and capable of contributing to the development of glaucomatous optic neuropathy, even without considering the probable accentuating effect of the lamina cribrosa's microarchitecture. Depending on optic cup shape, IOP-induced deformation of the vitreoretinal interface may not match lamina cribrosa deformation. This finding implies that scanning laser tomography has limited ability to estimate lamina cribrosa deformation when imaging the anterior topography of the optic nerve head. Biomechanical effects in the lamina cribrosa depend strongly on scleral properties.

Purpose: To investigate the efficacy of two different methods of adenoviral transfer of genes to trabecular meshwork (TM) and Schlemm's canal (SC) cells in cultured human anterior segments, using both experimental and numerical analyses., Methods: Replication-deficient adenoviruses having coding sequence for beta-galactosidase (beta-gal) under the control of the cytomegalovirus promoter were used. Efficiency of gene transfer over time was verified by infecting cultured human TM cells and assaying for beta-gal activity. Next, ostensibly normal paired human eyes were prepared by standard techniques and perfused for 2 to 5 days to measure baseline facilities. Eyes were then infected by one of two methods: standard transcorneal puncture, or injection into a 1 mm diameter silastic segment of supply tubing immediately upstream of the perfusion dish. In both cases, the nominal total dose was 2 x 10(8) viral particles. Five days after viral injection, eyes were harvested and fixed, and wedges from each of four quadrants were examined histologically. Sections were assayed for beta-gal activity and/or stained with toluidine blue. In a parallel study, flow and viral transport within perfused anterior segments were numerically simulated for conditions that approximated those used experimentally., Results: Eyes receiving viral particles by transcorneal injection showed variable levels of beta-gal activity and highly variable TM cellular morphology, ranging from excellent preservation to cellular lysis. Eyes receiving an equivalent viral dose via the supply tubing showed higher transfer efficiency, as judged by almost complete TM cell loss (indicative of viral toxicity) and intense extracellular beta-gal activity from the residual cytoplasm. At lower doses (1/3 to 1/1000 of that used in transcorneal injection) beta-gal activity was still present, while TM cell morphology was good at the lower viral doses. Computer modeling showed that the region beneath the cornea was nearly stagnant, and consequently virus introduced into this region by transcorneal injection was delivered very slowly to the TM. This caused the effective delivered viral dose to be low and sensitively dependent on the volume and shape of the transcorneally injected virus bolus., Conclusions: Injection of adenovirus into supply tubing led to consistent delivery of reporter gene and approximately 300-fold greater efficiency of gene transfer compared to the transcorneal injection method, and is therefore the preferred method for introducing viral particles into perfused anterior segments. These findings were consistent with computer modeling of flow and mass transport in perfused anterior segments. Although these quantitative results are specific to adenovirus, this general trend should hold for a wide range of perfused compounds.

Purpose: TGF-beta2 is known to be present at elevated levels in the aqueous humor of patients with primary open-angle glaucoma (POAG). Studies have shown that TGF-beta2 influences cultured trabecular meshwork (TM) cells, but the effects of this cytokine on intact TM and outflow facility have not been studied. The purpose of this study was to investigate whether TGF-beta2 treatment induces changes in outflow facility and morphologic changes in the TM tissue and whether these changes are comparable to those previously recorded in glaucomatous eyes., Methods: Baseline facility was measured in paired human eyes (n = 8 pairs), with a constant-flow anterior segment culture system. Medium perfusing experimental eyes was then supplemented with activated human recombinant TGF-beta2 (3.0 ng/mL, comparable to or slightly greater than measured aqueous humor levels in patients with POAG), and facility was measured for at least 8 days. At the conclusion of the perfusion, eyes were fixed and processed for light microscopy, transmission electron microscopy, and immunolabeling studies., Results: TGF-beta2 perfusion reduced outflow facility by 27% (P = 0.03) and promoted focal accumulation of fine fibrillar extracellular material in multilayered structures under the inner wall of Schlemm's canal. In treated eyes, Schlemm's canal was 27% shorter (P = 0.02), and the length of the inner wall apparently available for fluid flow was 33% less (P = 0.001), both compared with paired control eyes., Conclusions: TGF-beta2 reduces outflow facility when perfused into cultured human anterior segments. Furthermore, TGF-beta2 affects the extracellular matrix of the trabecular meshwork in a manner that is consistent with the observed reduction in outflow facility. Although the distribution of accumulated fibrillar material was different in these perfused eyes than that in POAG, the difference could be due to variation in biomechanical environment for TM cells in cultured anterior segments compared with the living eye. Overall, these results support the hypothesis that elevated TGF-beta2 levels in the aqueous humor play a role in the pathogenesis of the ocular hypertension in POAG.

Purpose: Mutations in the MYOC gene coding for myocilin are associated with elevated intraocular pressure (IOP), and recombinant myocilin, produced in a prokaryotic expression system, has been reported to affect aqueous outflow facility. This study was conducted to test whether perfusion with a fragment of recombinant myocilin (containing the full-length olfactomedin domain), produced in a eukaryotic expression system, affects facility., Methods: 293 EBNA cells were transfected by a vector containing the BM40 signal peptide, a human cDNA coding for myocilin, and a polyhistidine tag (HisTag) sequence. Recombinant protein was isolated by Ni-chelate chromatography, and characterized, and perfused into cultured anterior segments of human and porcine eyes., Results: Recombinant myocilin was secreted as a approximately 55-kDa intact protein and two fragments arising from cleavage of the recombinant protein at amino acid 215. The C-terminal fragment, containing the entire olfactomedin domain, was successfully isolated. When perfused into human and porcine eyes, this C-terminal fragment did not appreciably affect outflow facility., Conclusions: Although the olfactomedin domain appears to be important for the function of myocilin, perfusion with a recombinant myocilin fragment containing this domain does not change outflow facility. It is possible that both the olfactomedin and N-terminal domains (including the leucine zipper) must be present for myocilin to have full function. Alternatively, posttranslational modifications of myocilin may have a major impact on protein function.

Purpose: In a prior study, it has been reported that glaucomatous eyes have a significantly lower density of pores in the inner wall of Schlemm's canal than do normal eyes. However, in that study the glaucomatous eyes were fixed at much lower flow rates than the normal eyes, and that is now known to affect inner wall pore density. The objective of the current study was to compare the inner wall's pore density in glaucomatous and normal eyes, accounting for the effects of fixation conditions., Methods: Outflow facility was measured in enucleated glaucomatous human eyes. Eyes were fixed under constant flow conditions, microdissected to expose the inner wall of Schlemm's canal, and prepared for scanning electron microscopy (SEM). The density and diameter of the two subpopulations of pores in the inner wall, intracellular and intercellular (or "border") pores, were measured. Data were compared with those in previous studies of normal eyes., Results: As previously reported, pore density decreased with increasing postmortem time and increased with increasing volume of fixative passed through the outflow pathway and with increasing fixation time. Linear regression analysis indicated that glaucomatous eyes had less than one fifth the number of pores than normal eyes have, after accounting for the influence of volume of fixative perfused through the eyes (835 pores/mm(2) in normal eyes versus 160 pores/mm(2) in glaucomatous eyes). A nonlinear regression of pore density versus fixative volume produced a pore density at zero fixative volume that was not statistically different from zero. If true, this implies that all (or nearly all) inner wall pores observed by SEM are fixation artifacts. The density of intracellular pores and the diameter of these pores correlated with the density and diameter of the border pores, respectively., Conclusions: Inner wall pores are reduced in glaucomatous eyes. If pores are physiological structures, the elevated intraocular pressure characteristic of glaucoma may be explained by decreased porosity of the inner wall endothelium. Both border and intracellular pores seem to be induced in a similar fashion by fixation. The unlikely possibility that all inner wall pores are fixation-induced cannot be excluded. If so, a fundamental reassessment of the mechanism by which aqueous humor crosses the inner wall endothelium is necessary.

Purpose: To determine the effect of charged moieties within the outflow pathway on aqueous outflow facility in human eyes., Methods: After baseline facility measurement in human eye bank eyes (n = 10 pairs), one eye of each pair received anterior chamber exchange and continued perfusion with medium containing 10 mg/ml cationic ferritin. Contralateral eyes were treated in a similar manner with anionic ferritin (10.0 or 102 mg/ml). Eyes were fixed by anterior chamber exchange and perfusion with universal fixative at 8 mm Hg (corresponding to a physiologic pressure of 15 mm Hg in vivo) and examined by transmission electron microscopy. In a second series of human eyes (n = 8 pairs), facility was measured before and after anterior chamber exchange, with a solution containing 0.1 U/ml neuraminidase., Results: Perfusion of eyes with anionic ferritin at either 10.0 or 102 mg/ml caused a negligible 2% increase in facility, whereas cationic ferritin perfusion reduced facility by 66% (P < 0.00001). Perfusion with fixative reduced facility by approximately 60% in both cationic and anionic ferritin-perfused eyes, relative to facilities after perfusion with ferritin. Transmission electron microscopy showed that the distribution of ferritin was segmentally variable. Cationic ferritin consistently labeled the luminal surface of the inner wall of Schlemm's canal, and variably labeled the juxtacanalicular connective tissue (JCT) and trabecular beam surfaces. Anionic ferritin was more prominent in the JCT and intertrabecular spaces and less so on the luminal surface of Schlemm's canal. By scanning electron microscopy, cationic ferritin was seen to accumulate at intercellular margins of the inner wall. Neuraminidase perfusion had no significant effect on outflow facility., Conclusions: Cationic ferritin reduces outflow facility, presumably by binding to negatively charged sites in the outflow pathway. A possible mechanism is partial or complete blockage of intercellular clefts in the inner wall of Schlemm's canal by the ferritin that accumulates on the luminal surface of the inner wall. Although they are possible targets for ferritin binding, sialyl residues themselves seem to have little direct effect on outflow facility. Our data indicate that positively charged molecules, especially if they can interact with inner wall pores, have the potential to markedly alter outflow facility.

Purpose: The role of the inner wall of Schlemm's canal in determining aqueous outflow facility is poorly understood. To quantify the relationship between inner wall pore characteristics and aqueous outflow facility in human eyes, both control eyes and eyes in which facility had been pharmacologically increased by ethacrynic acid (ECA) infusion were studied., Methods: Outflow facility was measured in enucleated human eyes before and after delivery of 0.25 mM ECA (one eye of each of 6 pairs) or 2.5 mM ECA (one eye of each of 13 pairs). ECA, and vehicle in contralateral eyes, was delivered into Schlemm's canal by retroperfusion, thereby largely avoiding drug exposure to the trabecular meshwork. After facility measurement, eyes were fixed under conditions of either constant pressure (physiological intraocular pressure, 13 pairs) or "equal flow" (6 pairs) and were microdissected to expose the inner wall of Schlemm's canal. The density and diameter of intercellular and intracellular inner wall pores were measured using scanning electron microscopy., Results: Retroperfusion with 2.5 mM ECA increased facility by 73% (P < 0.001), whereas 0.25 mM ECA increased facility by 19% (not statistically significant). The density of intercellular pores in the inner wall of Schlemm's canal was increased by 520% in 2.5 mM ECA-retroperfused eyes (P < 0.00004), whereas intracellular pore density remained approximately constant. Large pores (size > or = 1.1 microm) were particularly enhanced in ECA retroperfused eyes. The net change in facility due to ECA was not correlated with changes in pore density or other inner wall pore statistics., Conclusions: Our data are most consistent with a model in which pores in the inner wall of Schlemm's canal indirectly influence facility. However, measured changes in facility due to changes in inner wall properties did not agree with quantitative predictions of the pore funneling theory, suggesting that changes in facility may instead be due to gel leakage from the extracellular spaces of the juxtacanalicular tissue. More definitive experiments are required to confirm this hypothesis.

Purpose: The influence of the inner wall of Schlemm's canal on aqueous outflow facility remains poorly understood. We examined the relationship between inner wall pore characteristics and outflow facility in living primate eyes in which facility had been pharmacologically increased by ethacrynic acid (ECA) infusion and in contralateral control eyes., Methods: Outflow facility (two-level constant pressure perfusion) was measured in eight pairs of living monkey eyes before and after administration of a bolus dose of either 0.125 mM ECA or vehicle. After exsanguination, eyes were fixed in situ under constant-pressure conditions (mean fixation pressure approximately 19 mm Hg). The density and diameter of inner wall pores and the number and area of platelet aggregates on the inner wall of Schlemm's canal were measured by scanning electron microscopy., Results: In ECA-treated eyes, outflow facility increased 63% (P < 0.0001), intracellular pore density decreased 46% (P = 0.0094), intracellular pore size increased 27% (P = 0.049), platelet aggregate density increased 158% (P < 0.0001), and area covered by platelets increased 210% (P = 0.012) relative to contralateral controls. Although the average density and size of intercellular pores were essentially unaffected by ECA, an increased density of large (> or = 1.90 microm) intercellular pores was seen in ECA-treated eyes. The density of intracellular pores increased with the duration of fixative perfusion. Other than a weak negative correlation between outflow facility and intracellular pore density in ECA-treated eyes (P = 0.052), facility was not correlated with inner wall pore features., Conclusions: Our data are most consistent with a scenario in which ECA promotes formation of large intercellular pores in the inner wall of Schlemm's canal, which are then masked by platelet aggregates. Masking of intercellular pores, combined with fixation-induced alteration of inner wall pore density, greatly complicates attempts to relate facility to inner wall structure and suggests that in vivo pore density is smaller than in fixed tissue. Additionally, facility-influencing effects of ECA on the juxtacanalicular tissue cannot be excluded.

Purpose: It has been reported that fixation conditions significantly influence the apparent pore density in the inner-wall endothelium of Schlemm's canal. In the present study, the manner in which fixation conditions affect the two subtypes of inner-wall pores, intracellular pores and intercellular (or border) pores, was investigated., Methods: Outflow facility was measured in enucleated human eyes. Eyes were fixed under constant flow" or constant pressure conditions, microdissected to expose the inner wall of Schlemm's canal, and prepared for scanning electron microscopy. The density and diameter of the two subtypes of pores in the inner wall were measured., Results: Intracellular pore density decreased with increasing postmortem time (P < 0.001) and increased with increasing volume of fixative passed through the outflow pathway (P < 0.001), whereas border pore density showed no dependence on these parameters (P > 0.25 and P > 0.15, respectively). Border pore density increased with increasing fixation pressure (P < 0.005), even though intracellular pore density showed no such dependence (P > 0.4). No correlation was found between outflow facility and the predictions of Poiseuille's law, Sampson's law, or the funneling theory for the hydraulic conductivity of the intracellular pores (P > 0.35) or the border pores (P > 0.1)., Conclusions: The intracellular and border pores form two morphologically and functionally distinct populations in the inner wall of Schlemm's canal. The dependence of intracellular pore density on postmortem time and on volume of fixative passed through the outflow pathway suggests that these pores are artifacts of tissue fixation or processing conditions. That border pores do not depend on such conditions and that their presence is correlative with perfusion pressure suggests that this population may be nonartifactual. New histologic techniques for examining the inner wall of Schlemm's canal are necessary to determine the in vivo state of inner-wall pores and how they influence outflow facility.

Purpose: A linear relationship between the density of pores in the inner wall of Schlemm's canal and aqueous outflow facility has been reported previously in a study in which investigators examined only eyes fixed at constant pressure, so that fixative flow rates differed from eye to eye. Because pores may form as a function of flow rate, the purpose in the current study was to verify the previous findings, using constant flow perfusions., Methods: Outflow facility was measured in enucleated human eyes. Eyes were fixed under either constant flow or constant pressure conditions, microdissected to expose the inner wall of Schlemm's canal, and prepared for scanning electron microscopy. The density and diameter of pores in the inner wall were measured., Results: Statistical analysis showed no correlation between outflow facility and either the density or the diameter of pores. Pore density decreased significantly during the hours after death. Examining only eyes for which experimentation was started within 20 hours of death, we found that pore density increased significantly with the volume of fixative that had been perfused through the outflow pathway., Conclusions: The correlation found by Allingham et al between outflow facility and pore density in the inner wall endothelium was not confirmed. However, the relationship between pore density and volume of fixative perfused is consistent with and may be responsible for the finding in the previous study. Because fixation conditions can influence the apparent pore density in the inner wall endothelium significantly, the conclusion reached previously, that pores contribute only 10% of the aqueous outflow resistance, may require reevaluation.

Purpose: To extend the retroperfusion technique to allow the delivery of drugs into Schlemm's canal in enucleated human eyes and to use this technique to gain insights into the function of the inner wall of Schlemm's canal., Methods: Using our previously developed retroperfusion technique, the anterior chamber of enucleated human eyes was held at a small negative pressure (-0.75 mm Hg), and fluid was allowed to flow retrograde from the limbal vessels, through the collector channels, and into Schlemm's canal. In this manner, the sulfhydryl agent N-ethyl maleimide (NEM) or the fixative agent glutaraldehyde was delivered to the inner wall of Schlemm's canal in normal and glaucomatous human eyes. Facility changes caused by retroperfusion were measured and correlated with histologic studies of the inner wall of Schlemm's canal., Results: Retroperfusion effectively delivers fluid from the scleral surface into the lumen of Schlemm's canal. Retroperfusion with vehicle alone does not alter facility or change outflow pathway morphology. Retroperfusion with NEM causes an approximately 35% facility increase and concomitant inner wall openings. Retroperfusion with glutaraldehyde in normal eyes and eyes with primary open-angle glaucoma causes a facility decrease of 53% and 64%, respectively, and localized fixation of the inner wall of Schlemm's canal. The magnitude of the facility changes caused by retroperfusion were similar to those seen using conventional forward perfusion of NEM and glutaraldehyde., Conclusions: Retroperfusion is a viable technique for the delivery of drugs or other agents into Schlemm's canal in enucleated human eyes. Retroperfusion-induced changes in outflow facility are correlated strongly with morphologically observed alterations in inner wall structure. The majority of outflow resistance is localized to the inner wall of Schlemm's canal or the immediately adjacent 10-microns region of the juxtacanalicular tissue in normal eyes and in eyes with primary open-angle glaucoma. Inner wall giant vacuoles and pores likely persist for sometime, even after fixation at zero or negative pressure.

Purpose: The goal of this study was to develop a new technique to deliver drugs or other agents to the lumen of the angular aqueous plexus/Schlemm's canal (AAP/SC) while bypassing the trabecular meshwork, thereby gaining insight into AAP/SC inner wall function., Methods: The anterior chamber is held at a small negative pressure and fluid is allowed to flow retrograde from the limbal vessels, through the collector channels, and into the AAP/SC ("retroperfusion"). Facility measurements are combined with histologic and tracer studies in bovine eyes., Results: (1) Retroperfusion with a saline solution does not alter facility or change outflow pathway morphology; (2) fluid is able to move retrograde from the scleral surface and enter the lumen of the AAP; and (3) retroperfusion with N-ethyl maleimide causes a dose-dependent increase in washout rate and concomitant inner wall breaks., Conclusions: It is hypothesized that the observed increase in washout is due to leakage of extracellular materials through breaks in the inner wall.

The inner wall (IW) endothelial lining of Schlemm's canal was examined in six normal human eyes and four eyes with primary open angle glaucoma (POAG). Outflow facility was measured using constant pressure perfusion at 15 mmHg, eyes were fixed at 15 mmHg, and the IW endothelial lining was isolated and examined by scanning electron microscopy. Pore density, pore diameter, and bulge density were recorded by quadrant, and pore size and density were used to estimate IW endothelial facility, resistance, and hydraulic conductivity (facility per unit area). In POAG eyes, pores were less common (489 +/- 172 vs 1437 +/- 423 pores/mm2; P less than .005) and appeared to be more unevenly distributed than in normal eyes. A regional analysis of pore density (by quadrant) failed to detect a significant difference between quadrants of normal or POAG eyes. Pore density was correlated with measured outflow facility in normal eyes alone (P less than .02) and when normal eyes were pooled with POAG eyes (P less than .001). The percentage of total resistance attributed to the IW endothelium was 5.8% in normals compared to 9.5% in POAG eyes. This indicates there is a greater pressure drop across the IW endothelium in POAG eyes, suggesting that an intrinsic difference in IW endothelial function exists between normal and glaucomatous eyes. However, this difference alone does not account for the decreased outflow facility in POAG eyes. IW endothelial hydraulic conductivity is markedly higher than that of other vascular endothelia. We hypothesize that this may protect the IW endothelial lining of Schlemm's canal from mechanical stress induced by the relatively high rate of transcellular fluid flow.

The juxtacanalicular connective tissue (JCT) is widely believed to generate the bulk of aqueous humor outflow resistance, while the pores of the inner wall endothelium are thought to generate at most 10% of this resistance in humans. However, the hydrodynamic interaction of these two components of the aqueous outflow system, which arises because of their spatial proximity, has only recently been considered. Modelling the JCT as a homogeneously distributed porous material upstream of a low porosity filter (the inner wall endothelium), the pores of the inner wall are found to cause a "funneling effect," in which the aqueous humor flows preferentially through those regions of the JCT nearest the inner wall pores. The bulk of the pressure drop occurs in the immediate proximity of the pores (within three pore radii). This greatly increases the apparent flow resistance of the JCT. For a set of parameters characterizing the normal eye, this enhancement is approximately 30-fold. The conclusion of this study is that changes in inner wall porosity may greatly affect aqueous outflow resistance, despite the low flow resistance of the inner wall pores themselves.

Previous studies show that hydrogen peroxide (H2O2) is present in the aqueous humor of many species and is capable of affecting outflow facility in animal model experiments. To study the hypothesis that oxidative damage to the outflow pathway may play a role in the pathogenesis of primary open-angle glaucoma, 3 mM H2O2 with 20 mM 3-aminotriazole and 1 mM carmustine (BCNU) in Dulbecco's phosphate-buffered saline (DPBS) was perfused into enucleated pig eyes at constant pressure. Baseline and experimental perfusions were done at two different pressures (7.5 and 30 mm Hg) to study the effect of pressure on the response to oxidative damage. Outflow facility in the baseline experiments (with DPBS only) was observed to increase nonlinearly with time during the perfusions, but could be linearized if plotted as a function of the volume perfused. Thus, a term "volumetric washout" (W) was introduced and defined as the fractional rate of change of outflow facility with respect to the volume perfused. This quantity was found to be independent of pressure in the baseline studies. Perfusion of H2O2 and inhibitors increased W at 7.5 mm Hg but decreased W at 30 mm Hg. These results indicate that oxidative damage increases outflow facility at normal pressure but decreases it at elevated pressure, suggesting that elevated pressure may increase the susceptibility of the outflow pathway to this form of insult.

It has recently been shown that aqueous humor is able to obstruct flow through Nuclepore polycarbonate filters having flow dimensions similar to those found in the juxtacanalicular tissue (JCT). We undertook studies designed to identify the component(s) of aqueous humor responsible for this obstruction and to determine the mechanism of blockage. We conclude that aqueous humor contains two components (one of which is specific to aqueous) which, when simultaneously present, hydrophobically bind to microporous filters and lead to filter blockage. Some implications for aqueous humor flow through the JCT are briefly discussed.

Flow resistance was measured as bovine and primate aqueous humor was passed through Nuclepore polycarbonate filters having flow dimensions similar to those found within the juxtacanalicular meshwork of the aqueous outflow network. The results indicate that aqueous humor has a greater flow resistance than isotonic saline; this greater resistance is attributable to proteins or glycoproteins in aqueous humor that obstruct the filters. If the same phenomenon is operative in the aqueous outflow network, it would help to explain discrepancies between calculated and measured aqueous outflow resistance.

The structure of the juxtacanalicular meshwork (JCM) was analyzed morphometrically, and the resulting data were used to calculate the resistance to flow through this tissue. Two models of the JCM were presented and compared. In the first (Model A), aqueous humor was assumed to flow via open channels within a solid framework, while, in the second (Model B), these open spaces were assumed to be filled with extracellular matrix gel. An expression giving the resistance of such a gel as a function of gel concentration was presented and tested on corneal and scleral stroma. Morphometry of normal and glaucomatous human eyes showed that Model A underpredicted the resistance of the JCM by factors of 10-100, suggesting that a GAG or proteoglycan gel may control the flow resistance of this tissue. This was supported by Model B, which showed that measured bulk concentrations of GAGs were consistent with gel concentrations needed to account for the estimated resistance of the JCM in vivo. Some limitations and implications of Model B were discussed.

Recent studies have demonstrated that bovine and primate aqueous humor (AH) obstruct flow when perfused through artificial membranes with pore sizes similar to those found in the aqueous outflow pathway. Proteinaceous AH components were implicated in this phenomenon, which is not observed with serum diluted to comparable protein concentrations. In this study, we used two-dimensional gel electrophoresis to characterize the protein composition of calf AH and to identify those proteins binding to the filters and presumably causing this obstruction. Comparison of AH and serum under denaturing conditions showed quantitative and qualitative differences in their protein content. Among the most important: AH was seen to possess two protein subunit trains (approximately 28 kD and approximately 48 kD) not found in serum and two trains (approximately 28 kD and approximately 80 kD) with additionally charged components not found in serum. Serum, on the other hand, possesses one train (approximately 80-90 kD) not found in AH as well as a slightly greater relative amount of high-molecular weight protein subunits. The finding that hydrophobic filters retain more protein components than do hydrophilic filters suggests that the type and amount of protein adhering to them is determined largely by hydrophobic interactions. Whether such interactions occur in the outflow system, and if so, how they may relate to aqueous drainage remains to be determined.