VEGF-A-induced changes in distal outflow tract structure and function.

PURPOSE: To investigate changes in distal outflow tract vessels caused by VEGF-A and their impact on outflow. METHODS: We compared VEGF-A perfused porcine anterior segments with and without trabecular meshwork (TM) to control eyes. In the first experiment (n=48), we analyzed live changes of the outflow tract with spectral-domain optical coherence tomography (SD-OCT) over 3 h and reconstructed them in 3D. In a second experiment (n=32), we measured the intraocular pressure (IOP) variation in response to VEGF-A over 48 h and computed the outflow facility. RESULTS: VEGF-A increased the vessel volume of the distal outflow tract by 16.8±10.6% while control eyes remained unchanged (0.5±6.8%). Volume changes occurred within the first 100 min before plateauing at 140 min. VEGF-A enhanced the outflow facility in eyes without TM by 38.6±25.5% at 24 h as compared to controls (p<0.05). CONCLUSION: VEGF-A dilated vessels of the distal outflow tract and increased the outflow facility even after TM removal, pointing to a regulatory mechanism independent of proximal structures.

Molecular taxonomy of human ocular outflow tissues defined by single-cell transcriptomics.

The conventional outflow pathway is a complex tissue responsible for maintaining intraocular pressure (IOP) homeostasis. The coordinated effort of multiple cells with differing responsibilities ensures healthy outflow function and IOP maintenance. Dysfunction of one or more resident cell types results in ocular hypertension and risk for glaucoma, a leading cause of blindness. In this study, single-cell RNA sequencing was performed to generate a comprehensive cell atlas of human conventional outflow tissues. We obtained expression profiles of 17,757 genes from 8,758 cells from eight eyes of human donors representing the outflow cell transcriptome. Upon clustering analysis, 12 distinct cell types were identified, and region-specific expression of candidate genes was mapped in human tissues. Significantly, we identified two distinct expression patterns (myofibroblast- and fibroblast-like) from cells located in the trabecular meshwork (TM), the primary structural component of the conventional outflow pathway. We also located Schwann cell and macrophage signatures in the TM. The second primary component structure, Schlemm's canal, displayed a unique combination of lymphatic/blood vascular gene expression. Other expression clusters corresponded to cells from neighboring tissues, predominantly in the ciliary muscle/scleral spur, which together correspond to the uveoscleral outflow pathway. Importantly, the utility of our atlas was demonstrated by mapping glaucoma-relevant genes to outflow cell clusters. Our study provides a comprehensive molecular and cellular classification of conventional and unconventional outflow pathway structures responsible for IOP homeostasis.

Measurement of postmortem outflow facility using iPerfusion.

The key risk factor for glaucoma is elevation of intraocular pressure (IOP) and alleviating it is the only effective therapeutic approach to inhibit further vision loss. IOP is regulated by the flow of aqueous humour across resistive tissues, and a reduction in outflow facility, is responsible for the IOP elevation in glaucoma. Measurement of outflow facility is therefore important when investigating the pathophysiology of glaucoma and testing candidate treatments for lowering IOP. Due to similar anatomy and response to pharmacological treatments, mouse eyes are a common model of human aqueous humour dynamics. The ex vivo preparation, in which an enucleated mouse eye is mounted in a temperature controlled bath and cannulated, has been well characterised and is widely used. The postmortem in situ model, in which the eyes are perfused within the cadaver, has received relatively little attention. In this study, we investigate the postmortem in situ model using the iPerfusion system, with a particular focus on i) the presence or absence of pressure-independent flow, ii) the effect of evaporation on measured flow rates and iii) the magnitude and pressure dependence of outflow facility and how these properties are affected by postmortem changes. Measurements immediately after cannulation and following multi-pressure facility measurement demonstrated negligible pressure-independent flow in postmortem eyes, in contrast to assumptions made in previous studies. Using a humidity chamber, we investigated whether the humidity of the surrounding air would influence measured flow rates. We found that at room levels of humidity, evaporation of saline droplets on the eye resulted in artefactual flow rates with a magnitude comparable to outflow, which were eliminated by a high relative humidity (>85%) environment. Average postmortem outflow facility was ∼4 nl/min/mmHg, similar to values observed ex vivo, irrespective of whether a postmortem delay was introduced prior to cannulation. The intra-animal variability of measured outflow facility values was also reduced relative to previous ex vivo data. The pressure-dependence of outflow facility was reduced in the postmortem relative to ex vivo model, and practically eliminated when eyes were cannulated >40 min after euthanisation. Overall, our results indicate that the moderately increased technical complexity associated with postmortem perfusion provides reduced variability and reduced pressure-dependence in outflow facility, when experimental conditions are properly controlled.

Decreased outflow facility and Schlemm's canal defects in a mouse model of glaucoma.

Previously we identified B6.EDA+/+ mice as a novel mouse model that presents with elevated IOP and trabecular meshwork damage. Here, we expand on our previous findings by measuring aqueous humor outflow facility and analyzing the integrity of the inner wall of Schlemm's canal. As expected, intraocular pressure (IOP) was increased, and outflow facility was decreased compared to C57BL/6J controls. B6.EDA+/+ mice had significantly increased expression of the adherens junction protein, VE-cadherin by the inner wall endothelium of Schlemm's canal. These data suggest that in addition to trabecular meshwork damage, there are changes in Schlemm's canal in B6.EDA+/+ mice that lead to aqueous outflow dysfunction and ocular hypertension.

Morphological changes to Schlemm's canal and the distal aqueous outflow pathway in monkey eyes with laser-induced ocular hypertension.

Though roughly 30-50% of aqueous outflow resistance resides distal to Schlemm's canal (SC), the morphology of the conventional outflow pathway distal to SC has not been thoroughly evaluated. This study examined the morphological changes along proximal and distal aspects of the conventional aqueous outflow pathway and their association with decreased outflow facility in an experimental model of glaucoma in cynomolgus macaques. Nd:YAG laser burns were made to 270-340 degrees of the trabecular meshwork (TM) of one eye (n = 6) or both eyes (n = 2) of each monkey to induce ocular hypertension. Distinct regions of the TM were left unlasered. Contralateral eyes (n = 5) were not lasered and were utilized as controls. Monkeys were sacrificed ≥58 months after their last laser treatment. All eyes were enucleated and perfused at 15 mmHg for 30 min to measure outflow facility. Two pairs of eyes were also perfused with fluorescein to examine segmental outflow. All eyes underwent perfusion-fixation for 1 h. Anterior segments were cut into radial wedges and processed for light and electron microscopy. Width, height, and cross-sectional area (CSA) of SC were compared between high- and low-flow regions of control eyes, and between non-lasered regions of laser-treated eyes and control eyes. Number and CSA of intrascleral veins (ISVs) were compared between non-lasered and lasered regions of laser-treated eyes and control eyes, and between high- and low-flow regions of control eyes. Scleral collagen fibril diameter was compared between control eyes and lasered and non-lasered regions of laser-treated eyes. Median outflow facility was significantly decreased in laser-treated eyes compared to control eyes (P = 0.02). Median CSA and height of SC were smaller in high-flow regions than low-flow regions of control eyes (P < 0.05). Median width of SC was not significantly different between high- and low-flow regions of control eyes (P > 0.05). Median CSA, width, and height of SC were not different between non-lasered regions and control eyes (P > 0.05). SC was partially or completely obliterated in lasered regions. Median number of ISVs was significantly decreased in lasered regions compared to non-lasered regions (P < 0.01) and control eyes (P < 0.01). Median CSA of ISVs did not differ between these groups (P > 0.05). Median number and CSA of ISVs were not significantly different between high- and low-flow regions of control eyes (P > 0.05). Lasered regions displayed looser scleral stroma and smaller median diameter of collagen fibrils adjacent to the TM compared to non-lasered regions (P < 0.05) and control eyes (P < 0.05). Dense TM, partial to complete obliteration of SC, and a decreased number of patent ISVs may account in part for the decreased outflow facility in monkey eyes with laser-induced ocular hypertension. The significance of changes in scleral structure in laser-treated eyes warrants further investigation.

Mechanistic Effects of Baicalein on Aqueous Humor Drainage and Intraocular Pressure.

Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that results from impeded fluid drainage. The increase in outflow resistance is caused by trabecular meshwork (TM) cell dysfunction and excessive extracellular matrix (ECM) deposition. Baicalein (Ba) is a natural flavonoid and has been shown to regulate cell contraction, fluid secretion, and ECM remodeling in various cell types, suggesting the potential significance of regulating outflow resistance and IOP. We demonstrated that Ba significantly lowered the IOP by about 5 mmHg in living mice. Consistent with that, Ba increased the outflow facility by up to 90% in enucleated mouse eyes. The effects of Ba on cell volume regulation and contractility were examined in primary human TM (hTM) cells. We found that Ba (1-100 µM) had no effect on cell volume under iso-osmotic conditions but inhibited the regulatory volume decrease (RVD) by up to 70% under hypotonic challenge. In addition, Ba relaxed hTM cells via reduced myosin light chain (MLC) phosphorylation. Using iTRAQ-based quantitative proteomics, 47 proteins were significantly regulated in hTM cells after a 3-h Ba treatment. Ba significantly increased the expression of cathepsin B by 1.51-fold and downregulated the expression of D-dopachrome decarboxylase and pre-B-cell leukemia transcription factor-interacting protein 1 with a fold-change of 0.58 and 0.40, respectively. We suggest that a Ba-mediated increase in outflow facility is triggered by cell relaxation via MLC phosphorylation along with inhibiting RVD in hTM cells. The Ba-mediated changes in protein expression support the notion of altered ECM homeostasis, potentially contributing to a reduction of outflow resistance and thereby IOP.

Thrombospondin-1 mediates Rho-kinase inhibitor-induced increase in outflow-facility.

Rho-kinase (ROCK) inhibitors, a novel class of anti-glaucoma agents, act by increasing the aqueous humor outflow through the conventional trabecular meshwork pathway. However, the downstream signaling consequences of the ROCK inhibitor are not completely understood. Our data show that Y39983, a selective ROCK inhibitor, could induce filamentous actin remodeling, reduced cell motility (as measured by cell migration), and transepithelial resistance in primary human TM (hTM) cells. After 2 days Y39983 treatment of hTM cells, a proteomic study identified 20 proteins whose expression was significantly altered. Pathway analysis of those proteins revealed the involvement of the p53 pathway, integrin signaling pathway, and cytoskeletal pathway regulation by Rho GTPase. Thrombospondin-1 (TSP1), a matricellular protein that is increased in glaucoma patients, was downregulated fivefold following Y39983 treatment. More importantly, both TSP1 antagonist leucine-serine-lysine-leucine (LSKL) and small interfering RNA (siRNA) reduced TSP1 gene and protein expressions as well as hTM cell migration. In the presence of Y39983, no further inhibition of cell migration resulted after LSKL and TSP1 siRNA knockdown. Likewise, LSKL triggered a dose-dependent increase in outflow facility in ex vivo mouse eyes, to a similar extent as Y39983 (83.8% increase by Y39983 vs. 71.2% increase by LSKL at 50 µM). There were no additive effects with simultaneous treatment with LSKL and Y39983, supporting the notion that the effects of ROCK inhibition were mediated by TSP1.

Intracranial pressure modulates aqueous humour dynamics of the eye.

KEY POINTS: An elevation in intracranial pressure (ICP) lowers conventional outflow facility (increases aqueous outflow resistance) of rat eyes. The reduction in outflow facility correlates with an increase in intraocular pressure (IOP). The effect of ICP elevation on outflow facility and IOP is blocked by TTX. The results indicate that aqueous humour dynamics is modulated by ICP-driven neural feedback from the brain. This feedback mechanism may act to stabilize translaminar pressure across the optic nerve head and may provide a new avenue for glaucoma therapy. ABSTRACT: While intraocular pressure (IOP) is a well-known risk factor for glaucoma, intracranial pressure (ICP) is attracting heightened interest because of its influence on optic nerve head biomechanics. Studies have shown that ICP can have marked impacts on posterior eye health by modifying the translaminar pressure gradient across the optic nerve. There is also growing evidence that IOP and ICP may be interconnected, although the mechanism of their putative interaction is unknown. We sought to test the hypothesis that ICP modulates IOP by altering aqueous humour dynamics. The anterior chamber and lateral ventricle of anaesthetized Brown-Norway rats were cannulated with fine-gauge needles connected to a programmable pump and saline reservoir, respectively. ICP was manipulated by varying reservoir height, and eye outflow facility (C) was determined from the pump flow rate required to hold IOP at different levels. C was 22 ± 4 nl/min/mmHg at resting ICP and 13 ± 3 nl/min/mmHg when ICP was raised 15 mmHg, a reduction of 41 ± 13% (n = 18). The decrease in outflow facility was independent of blood pressure, reversible, scaled with ICP elevation and correlated with increases in resting IOP. It was physiological in origin because C returned to baseline values after the rats were killed and corneal application of TTX though ICP remained elevated. These results indicate that a neural feedback mechanism driven by ICP regulates conventional outflow facility in rats. The mechanism may protect the eye from translaminar pressure swings and may offer a new target for glaucoma treatment.

Tissue plasminogen activator attenuates outflow facility reduction in mouse model of juvenile open angle glaucoma.

Tissue plasminogen activator (tPA) has been shown to prevent steroid-induced reduction in aqueous humor outflow facility via an upregulation in matrix metalloproteinase (Mmp) expression. The purpose of this study was to determine whether tPA can rescue outflow facility reduction in the Tg-MYOCY437H mouse model, which replicates human juvenile open angle glaucoma. Outflow facility was measured in Tg-MYOCY437H mice following: periocular steroid exposure and intraocular protein treatment with enzymatically active or enzymatically inactive tPA. Effects of tPA on outflow facility were compared to those of animals treated with topical sodium phenylbutarate (PBA), a modulator of endoplasmic reticulum stress. Gene expression of fibrinolytic pathway components (Plat, Plau, and Pai-1) and matrix metalloproteinases (Mmp-2, -9, and -13) was determined in angle ring tissues containing the trabecular meshwork. Tg-MYOCY437H mice did not display further outflow facility reduction following steroid exposure. Enzymatically active and enzymatically inactive tPA were equally effective in attenuating outflow facility reduction in Tg-MYOCY437H mice and caused enhanced expression of matrix metalloproteinases (Mmp-9 and Mmp-13). tPA was equally effective to topical PBA treatment in ameliorating outflow facility reduction in Tg-MYOCY437H mice. Both treatments were associated with an upregulation in Mmp-9 expression while tPA also upregulated Mmp-13 expression. tPA increases the expression of matrix metalloproteinases and may cause extracellular matrix remodeling at the trabecular meshwork, which results in reversal of outflow facility reduction in Tg-MYOCY437H mice.

Reduced humidity experienced by mice in vivo coincides with reduced outflow facility measured ex vivo.

Mice are routinely used to study aqueous humour dynamics. However, physical factors such as temperature and hydration affect outflow facility in enucleated eyes. This retrospective study examined whether differences in temperature and relative humidity experienced by living mice within their housing environment in vivo coincide with differences in outflow facility measured ex vivo. Facility data and environmental records were collected for one enucleated eye from 116 mice (C57BL/6J males, 9-15 weeks old) at two institutions. Outflow facility was reduced when relative humidity was below the lower limit of 45% recommended by the UK Code of Practice, but there was no detectable effect of temperature on outflow facility. Even when accounting for effects of humidity, there were differences in outflow facility measured between institutions and between individual researchers at the same institution. These data indicate that humidity, as well as additional environmental factors experienced by living mice within their housing environment, may significantly affect outflow facility measured ex vivo.

Outflow facility and extent of angle closure in a porcine model.

PURPOSE: To establish the extent of anterior chamber angle circumference needed to maintain a physiological outflow facility (C). This could create a model to investigate focal outflow regulation. METHODS: Twenty anterior segments of porcine eyes were assigned to five groups, each with a different degree of cyanoacrylate-mediated angle closure: 90° (n = 4), 180° (n = 4), 270° (n = 4), 360° (n = 4), and four unoccluded control eyes. The outflow facility was measured at baseline, 3, 12, 24, and 36 h after angle closure. Outflow patterns were evaluated with canalograms and the histomorphology was compared. RESULTS: Baseline outflow facilities of the five groups were similar (F = 0.922, p = 0.477). Occlusion of 360° induced a significant decrease in facility from baseline at all time-points (p ≤ 0.023 at 3, 12, 24, and 36 h). However, no difference from baseline was found in any of the partially occluded (0-270°) groups (F ≥ 0.067, p ≥ 0.296 at 3, 12, 24, and 36 h). The canalograms confirmed the extent of occlusion with flow through the unblocked regions. Histology revealed no adverse effects of blockage on the TM or aqueous plexus in the unoccluded angle portions. The unoccluded TM appeared normal. CONCLUSION: Cyanoacrylate-mediated angle occlusion created a reproducible angle closure model. Ninety degrees of unoccluded anterior chamber angle circumference was sufficient to maintain physiological outflow. This model may help understand how outflow can be regulated in healthy, nonglaucomatous TM.

Outflow enhancement by three different ab interno trabeculectomy procedures in a porcine anterior segment model.

PURPOSE: To evaluate three different microincisional ab interno trabeculectomy procedures in a porcine eye perfusion model. METHODS: In perfused porcine anterior segments, 90° of trabecular meshwork (TM) was ablated using the Trabectome (T; n = 8), Goniotome (G; n = 8), or Kahook device (K; n = 8). After 24 h, additional 90° of TM was removed. Intraocular pressure (IOP) and outflow facility were measured at 5 and 10 µl/min perfusion to simulate an elevated IOP. Structure and function were assessed with canalograms and histology. RESULTS: At 5 µl/min infusion rate, T resulted in a greater IOP reduction than G or K from baseline (76.12% decrease versus 48.19% and 47.96%, P = 0.013). IOP reduction between G and K was similar (P = 0.420). Removing another 90° of TM caused an additional IOP reduction only in T and G but not in K. Similarly, T resulted in the largest increase in outflow facility at 5 µl/min compared with G and K (first ablation, 3.41 times increase versus 1.95 and 1.87; second ablation, 4.60 versus 2.50 and 1.74) with similar results at 10 µl/min (first ablation, 3.28 versus 2.29 and 1.90 (P = 0.001); second ablation, 4.10 versus 3.01 and 2.01 (P = 0.001)). Canalograms indicated circumferential flow beyond the ablation endpoints. CONCLUSIONS: T, G, and K significantly increased the outflow facility. In this model, T had a larger effect than G and K.

Trabecular meshwork stiffness in glaucoma.

Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open-angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho-kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor-ß2 (TGF-ß2), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM-based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties.

Age-related posterior ciliary muscle restriction - A link between trabecular meshwork and optic nerve head pathophysiology.

The ciliary muscle plays a major role in controlling both accommodation and outflow facility in primates. The ciliary muscle and the choroid functionally form an elastic network that extends from the trabecular meshwork all the way to the back of the eye and ultimately attaches to the elastic fiber ring that surrounds the optic nerve and to the lamina cribrosa through which the nerve passes. The ciliary muscle governs the accommodative movement of the elastic network. With age ciliary muscle mobility is restricted by progressively inelastic posterior attachments and the posterior restriction makes the contraction progressively isometric; placing increased tension on the optic nerve region. In addition, outflow facility also declines with age and limbal corneoscleral contour bows inward. Age-related loss in muscle movement and altered limbal corneoscleral contour could both compromise the basal function of the trabecular meshwork. Further, recent studies in non-human primates show that the central vitreous moves posteriorly all the way back to the optic nerve region, suggesting a fluid current and a pressure gradient toward the optic nerve. Thus, there may be pressure and tension spikes on the optic nerve region during accommodation and these pressure and tension spikes may increase with age. This constellation of events could be relevant to glaucomatous optic neuropathy. In summary, our hypothesis is that glaucoma and presbyopia may be literally linked to each other, via the choroid, and that damage to the optic nerve may be inflicted by accommodative intraocular pressure and choroidal tension "spikes", which may increase with age.

Anterior chamber perfusion versus posterior chamber perfusion does not influence measurement of aqueous outflow facility in living mice by constant flow infusion.

Mice are now routinely utilized in studies of aqueous humor outflow dynamics. In particular, conventional aqueous outflow facility (C) is routinely measured via perfusion of the aqueous chamber by a number of laboratories. However, in mouse eyes perfused ex-vivo, values for C are variable depending upon whether the perfusate is introduced into the posterior chamber (PC) versus the anterior chamber (AC). Perfusion via the AC leads to posterior bowing of the iris, and traction on the iris root/scleral spur, which may increase C. Perfusion via the PC does not yield this effect. But the equivalent situation in living mice has not been investigated. We sought to determine whether AC versus PC perfusion of the living mouse eye may lead to different values for C. All experiments were conducted in C57BL/6J mice (all ♀) between the ages of 20 and 30 weeks. Mice were divided into groups of 3-4 animals each. In all groups, both eyes were perfused. C was measured in groups 1 and 2 by constant flow infusion (from a 50 µL microsyringe) via needle placement in the AC, and in the PC, respectively. To investigate the effect of ciliary muscle (CM) tone on C, groups 3 and 4 were perfused live via the AC or PC with tropicamide (muscarinic receptor antagonist) added to the perfusate at a concentration of 100 µM. To investigate immediate effect of euthanasia, groups 5 and 6 were perfused 15-30 min after death via the AC or PC. To investigate the effect of CM tone on C immediately following euthanasia, groups 7 and 8 were perfused 15-30 min after death via the AC or PC with tropicamide added to the perfusate at a concentration of 100 µM. C in Groups 1 (AC perfusion) and 2 (PC perfusion) was computed to be 19.5 ± 0.8 versus 21.0 ± 2.1 nL/min/mmHg, respectively (mean ± SEM, p > 0.4, not significantly different). In live animals in which tropicamide was present in the perfusate, C in Group 3 (AC perfusion) was significantly greater than C in Group 4 (PC perfusion) (22.0 ± 4.0 versus 14.0 ± 2.0 nL/min/mmHg, respectively, p = 0.0021). In animals immediately following death, C in groups 5 (AC perfusion) and 6 (PC perfusion) was computed to be 21.2 ± 2.0 versus 22.8 ± 1.4 nL/min/mmHg, respectively (mean ± SEM, p = 0.1196, not significantly different). In dead animals in which tropicamide was present in the perfusate, C in group 7 (AC perfusion) was greater than C in group 8 (PC perfusion) (20.6 ± 1.4 versus 14.2 ± 2.6 nL/min/mmHg, respectively, p < 0.0001). C in eyes in situ in living mice or euthanized animals within 15-30 min post mortem is not significantly different when measured via AC perfusion or PC perfusion. In eyes of live or freshly euthanized mice, C is greater when measured via AC versus PC perfusion when tropicamide (a mydriatic and cycloplegic agent) is present in the perfusate.

Direct measurement of pressure-independent aqueous humour flow using iPerfusion.

Reduction of intraocular pressure is the sole therapeutic target for glaucoma. Intraocular pressure is determined by the dynamics of aqueous humour secretion and outflow, which comprise several pressure-dependent and pressure-independent mechanisms. Accurately quantifying the components of aqueous humour dynamics is essential in understanding the pathology of glaucoma and the development of new treatments. To better characterise aqueous humour dynamics, we propose a method to directly measure pressure-independent aqueous humour flow. Using the iPerfusion system, we directly measure the flow into the eye when the pressure drop across the pressure-dependent pathways is eliminated. Using this approach we address i) the magnitude of pressure-independent flow in ex vivo eyes, ii) whether we can accurately measure an artificially imposed pressure-independent flow, and iii) whether the presence of a pressure-independent flow affects our ability to measure outflow facility. These studies are conducted in mice, which are a common animal model for aqueous humour dynamics. In eyes perfused with a single cannula, the average pressure-independent flow was 1 [-3, 5] nl/min (mean [95% confidence interval]) (N = 6). Paired ex vivo eyes were then cannulated with two needles, connecting the eye to both iPerfusion and a syringe pump, which was used to impose a known pressure-independent flow of 120 nl/min into the experimental eye only. The measured pressure-independent flow was then 121 [117, 125] nl/min (N = 7), indicating that the method could measure pressure-independent flow with high accuracy. Finally, we showed that the artificially imposed pressure-independent flow did not affect our ability to measure facility, provided that the pressure-dependence of facility and the true pressure-independent flow were accounted for. The present study provides a robust method for measurement of pressure-independent flow, and demonstrates the importance of accurately quantifying this parameter when investigating pressure-dependent flow or outflow facility.

Analysis of a method for establishing a model with more stable chronic glaucoma in rhesus monkeys.

In this study, we utilized yellow-wavelength laser treatment and measured aqueous outflow facility to establish a model for chronic glaucoma in rhesus monkeys. We then compared the effects of photocoagulation resulting from exposure to the yellow laser or to a green laser. Twelve rhesus monkeys were used to establish the model, and the yellow and green lasers were utilized for 360° photocoagulation in the anterior-chamber angles of the right eye in all subjects. After certain periods of time before and after the creation of the glaucoma model, the cornea, aqueous humor, optic cup, intraocular pressure (IOP), outflow facility, retinal nerve fiber layer (RNFL), and pathology of the trabecular meshwork were analyzed. Both the yellow and green lasers caused an increase in IOP compared with before photocoagulation (18.6 ± 2.6 mm Hg and 16.1 ± 1.8 mm Hg, respectively), with an average photocoagulation from the yellow and green lasers of 39.2 ± 7.9 mm Hg and 30.3 ± 4.7 mm Hg, respectively (P < 0.01). However, the success rate of a second photocoagulation treatment in the yellow laser group was significantly higher than in the green laser group (P < 0.05). After the increase in IOP, both groups exhibited an inflammatory response in the anterior segment, enlarged cupping, and a decrease in the average thickness of the RNFL. However, the yellow laser caused less corneal edema than the green laser (P < 0.05), and the outflow facility of the two groups (0.33 ± 0.09 and 0.30 ± 0.07 µl/min/mm Hg for the yellow and green lasers, respectively) showed different degrees of differences (0.05 ± 0.02 and 0.07 ± 0.02 µl/min/mm Hg for the yellow and green lasers, respectively) into the abnormal range after photocoagulation. Pathological examination revealed that the depth of destruction of the trabecular meshwork appeared to be deeper in the yellow laser group than in the green laser group. In conclusion, application of a yellow laser combined with measuring aqueous outflow facility produced a glaucoma model with a minor inflammatory response and few IOP fluctuations.

Aging and intraocular pressure homeostasis in mice.

Age and elevated intraocular pressure (IOP) are the two primary risk factors for glaucoma, an optic neuropathy that is the leading cause of irreversible blindness. In most people, IOP is tightly regulated over a lifetime by the conventional outflow tissues. However, the mechanistic contributions of age to conventional outflow dysregulation, elevated IOP and glaucoma are unknown. To address this gap in knowledge, we studied how age affects the morphology, biomechanical properties and function of conventional outflow tissues in C57BL/6 mice, which have an outflow system similar to humans. As reported in humans, we observed that IOP in mice was maintained within a tight range over their lifespan. Remarkably, despite a constellation of age-related changes to the conventional outflow tissues that would be expected to hinder aqueous drainage and impair homeostatic function (decreased cellularity, increased pigment accumulation, increased cellular senescence and increased stiffness), outflow facility, a measure of conventional outflow tissue fluid conductivity, was stable with age. We conclude that the murine conventional outflow system has significant functional reserve in healthy eyes. However, these age-related changes, when combined with other underlying factors, such as genetic susceptibility, are expected to increase risk for ocular hypertension and glaucoma.

TRPV4 subserves physiological and pathological elevations in intraocular pressure.

Ocular hypertension (OHT) caused by mechanical stress and chronic glucocorticoid exposure reduces the hydraulic permeability of the conventional outflow pathway. It increases the risk for irreversible vision loss, yet healthy individuals experience nightly intraocular pressure (IOP) elevations without adverse lifetime effects. It is not known which pressure sensors regulate physiological vs. pathological OHT nor how they impact the permeability of the principal drainage pathway through the trabecular meshwork (TM). We report that OHT induced by the circadian rhythm, occlusion of the iridocorneal angle and glucocorticoids requires activation of TRPV4, a stretch-activated cation channel. Wild-type mice responded to nocturnal topical administration of the agonist GSK1016790A with IOP lowering, while intracameral injection of the agonist elevated diurnal IOP. Microinjection of TRPV4 antagonists HC067047 and GSK2193874 lowered IOP during the nocturnal OHT phase and in hypertensive eyes treated with steroids or injection of polystyrene microbeads. Conventional outflow-specific Trpv4 knockdown induced partial IOP lowering in mice with occluded iridocorneal angle and protected retinal neurons from pressure injury. Indicating a central role for TRPV4-dependent mechanosensing in trabecular outflow, HC067047 doubled the outflow facility in TM-populated steroid-treated 3D nanoscaffolds. Tonic TRPV4 signaling thus represents a fundamental property of TM biology as a driver of increased in vitro and in vivo outflow resistance. The TRPV4-dependence of OHT under conditions that mimic primary and secondary glaucomas could be explored as a novel target for glaucoma treatments.

The Effect of Scleral Buckle Surgery on Tonographic Outflow Facility, Positional Intraocular Pressure, and Ocular Biomechanics.

PURPOSE: To investigate the in vivo effect of scleral buckle surgery on ocular biomechanics and aqueous humor dynamics. DESIGN: Prospective observational cross-sectional study. PARTICIPANTS: Nine patients with unilateral 360 degree encircling scleral buckles without vitrectomy for rhegmatogenous retinal detachments, between 3 and 39 months postoperative. METHODS: All measurements were performed in both eyes of all participants. Intraocular pressure (IOP) was measured in the seated and supine positions using pneumatonometry. Outflow facility was measured using 2-minute weighted pneumatonography. Ocular rigidity coefficient was determined from the Friedenwald equations based on the difference in IOP with and without a weighted tonometer tip. The percentage change in IOP upon transitioning from seated to supine was calculated. Measurements for buckled and nonbuckled eyes were compared using paired Student t test of means. MAIN OUTCOME MEASURES: Sitting and supine IOP and percentage difference between the 2 positions; outflow facility; ocular rigidity coefficient. RESULTS: Seated IOP was similar between buckled and nonbuckled eyes (16.1 ± 2.5 vs. 16.7 ± 2.7 mmHg; P = 0.5) whereas supine IOP was lower in buckled eyes compared with nonbuckled eyes (18.7 ± 2.6 vs. 21.3 ± 2.5 mmHg; P = 0.008). The percentage increase in IOP upon change in body position from seated to supine was greater in nonbuckled eyes (17.4 ± 9.4% vs. 27.6 ± 9.5%; P = 0.005). Ocular rigidity coefficient was lower in buckled (9.9 × 10-3 ± 1.4 × 10-3 µL-1) vs. nonbuckled eyes (14.4 × 10-3 ± 3.1 × 10-3 µL-1; P = 0.006). Outflow facility was not significantly different in buckled and nonbuckled eyes. CONCLUSIONS: Scleral buckling decreases ocular rigidity but does not affect outflow facility. This change in ocular biomechanics likely results in the attenuated IOP change from seated to supine position. Decreased ocular rigidity may also reduce IOP fluctuations and potentially reduce the risk for glaucoma progression. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.