Aqueous humor as eye lymph: A crossroad between venous and lymphatic system.

Aqueous humor (AQH) is a transparent fluid with characteristics similar to those of the interstitial fluid, which fills the eyeball posterior and anterior chambers and circulates in them from the sites of production to those of drainage. The AQH volume and pressure homeostasis is essential for the trophism of the ocular avascular tissues and their normal structure and function. Different AQH outflow pathways exist, including a main pathway, quite well defined anatomically and referred to as the conventional pathway, and some accessory pathways, more recently described and still not fully morphofunctionally understood, generically referred to as unconventional pathways. The conventional pathway is based on the existence of a series of conduits starting with the trabecular meshwork and Schlemm's Canal and continuing with a system of intrascleral and episcleral venules, which are tributaries to veins of the anterior segment of the eyeball. The unconventional pathways are mainly represented by the uveoscleral pathway, in which AQH flows through clefts, interstitial conduits located in the ciliary body and sclera, and then merges into the aforementioned intrascleral and episcleral venules. A further unconventional pathway, the lymphatic pathway, has been supported by the demonstration of lymphatic microvessels in the limbal sclera and, possibly, in the uvea (ciliary body, choroid) as well as by the ocular glymphatic channels, present in the neural retina and optic nerve. It follows that AQH may be drained from the eyeball through blood vessels (TM-SC pathway, US pathway) or lymphatic vessels (lymphatic pathway), and the different pathways may integrate or compensate for each other, optimizing the AQH drainage. The present review aims to define the state-of-the-art concerning the structural organization and the functional anatomy of all the AQH outflow pathways. Particular attention is paid to examining the regulatory mechanisms active in each of them. The new data on the anatomy and physiology of AQH outflow pathways is the key to understanding the pathophysiology of AQH outflow disorders and could open the way for novel approaches to their treatment.

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.

Impaired TRPV4-eNOS signaling in trabecular meshwork elevates intraocular pressure in glaucoma.

Primary Open Angle Glaucoma (POAG) is the most common form of glaucoma that leads to irreversible vision loss. Dysfunction of trabecular meshwork (TM) tissue, a major regulator of aqueous humor (AH) outflow resistance, is associated with intraocular pressure (IOP) elevation in POAG. However, the underlying pathological mechanisms of TM dysfunction in POAG remain elusive. In this regard, transient receptor potential vanilloid 4 (TRPV4) cation channels are known to be important Ca2+ entry pathways in multiple cell types. Here, we provide direct evidence supporting Ca2+ entry through TRPV4 channels in human TM cells and show that TRPV4 channels in TM cells can be activated by increased fluid flow/shear stress. TM-specific TRPV4 channel knockout in mice elevated IOP, supporting a crucial role for TRPV4 channels in IOP regulation. Pharmacological activation of TRPV4 channels in mouse eyes also improved AH outflow facility and lowered IOP. Importantly, TRPV4 channels activated endothelial nitric oxide synthase (eNOS) in TM cells, and loss of eNOS abrogated TRPV4-induced lowering of IOP. Remarkably, TRPV4-eNOS signaling was significantly more pronounced in TM cells compared to Schlemm's canal cells. Furthermore, glaucomatous human TM cells show impaired activity of TRPV4 channels and disrupted TRPV4-eNOS signaling. Flow/shear stress activation of TRPV4 channels and subsequent NO release were also impaired in glaucomatous primary human TM cells. Together, our studies demonstrate a central role for TRPV4-eNOS signaling in IOP regulation. Our results also provide evidence that impaired TRPV4 channel activity in TM cells contributes to TM dysfunction and elevated IOP in glaucoma.

Endothelial Tyrosine Kinase Tie1 Is Required for Normal Schlemm's Canal Development-Brief Report.

BACKGROUND: Schlemm's canal (SC) is a large vessel residing in the iridocorneal angle and is required to regulate aqueous humor outflow. Normal SC structure and function is indispensable for maintaining normal intraocular pressure, and elevated intraocular pressure is a risk factor for development of glaucoma. Recent reports have identified a key role of the angiopoietin-Tie2 pathway for SC development and function; however, the role of the orphan receptor Tie1 has not been clarified. METHODS: We used Tie1 knock out mice to study the function of Tie1 in SC development and function. Real-time quantitative polymerase chain reaction and Western blot analyses were used to verify Tie1 deletion. High-resolution microscopy of mouse SC whole mount and cross sections were used to study SC morphology. Measurement of intraocular pressure in live mice was used to study the impact of Tie1 on SC function. RESULTS: Tie1 is highly expressed in both human and mouse SC. Tie1 knock out mice display hypomorphic SC and elevated intraocular pressure as a result of attenuated SC development. CONCLUSIONS: Tie1 is indispensable for SC development and function, supporting it as a novel target for future SC-targeted glaucoma therapies and a candidate gene for glaucoma in humans.

Tissue-engineered anterior segment eye cultures demonstrate hallmarks of conventional organ culture.

BACKGROUND: Glaucoma is a blinding disease largely caused by dysregulation of outflow through the trabecular meshwork (TM), resulting in elevated intraocular pressure (IOP). We hypothesized that transplanting TM cells into a decellularized, tissue-engineered anterior segment eye culture could restore the outflow structure and function. METHODS: Porcine eyes were decellularized with freeze-thaw cycles and perfusion of surfactant. We seeded control scaffolds with CrFK cells transduced with lentiviral vectors to stably express eGFP and compared them to scaffolds seeded with primary TM cells as well as to normal, unaltered eyes. We tracked the repopulation behavior, performed IOP maintenance challenges, and analyzed the histology. RESULTS: Transplanted cells localized to the TM and progressively infiltrated the extracellular matrix, reaching a distribution comparable to normal, unaltered eyes. After a perfusion rate challenge to mimic a glaucomatous pressure elevation, transplanted and normal eyes reestablished a normal intraocular pressure (transplanted = 16.5 ± 0.9 mmHg, normal = 16.9 ± 0.9). However, eyes reseeded with eGFP-expressing CrFK cells could not regulate IOP, remaining high and unstable (27.0 ± 6.2 mmHg) instead. CONCLUSION: Tissue-engineered anterior segment scaffolds can serve as readily available, scalable ocular perfusion cultures. This could reduce dependency on scarce donor globes in outflow research and may allow engineering perfusion cultures with specific geno- and phenotypes.

Suberoylanilide hydroxamic acid (SAHA) inhibits transforming growth factor-beta 2-induced increases in aqueous humor outflow resistance.

Transforming growth factor-beta 2 (TGF-ß2) is highly concentrated in the aqueous humor of primary open-angle glaucoma patients. TGF-ß2 causes fibrosis of outflow tissues, such as the trabecular meshwork (TM), and increases intraocular pressure by increasing resistance to aqueous humor outflow. Recently, histone deacetylase (HDAC) activity was investigated in fibrosis in various tissues, revealing that HDAC inhibitors suppress tissue fibrosis. However, the effect of HDAC inhibitors on fibrosis in the eye was not determined. Here, we investigated the effect of suberoylanilide hydroxamic acid (SAHA), an HDAC inhibitor, on TGF-ß2-induced increased resistance to aqueous humor outflow. We found that SAHA suppressed TGF-ß2-induced outflow resistance in perfused porcine eyes. Moreover, SAHA cotreatment suppressed TGF-ß2-induced ocular hypertension in rabbits. The permeability of monkey TM (MTM) and Schlemm's canal (MSC) cell monolayers was decreased by TGF-ß2 treatment. SAHA inhibited the effects of TGF-ß2 on the permeability of these cells. TGF-ß2 also increased the expression of extracellular matrix proteins (fibronectin and collagen type I or IV) in MTM, MSC, and human TM (HTM) cells, while SAHA inhibited TGF-ß2-induced extracellular matrix protein expression in these cells. SAHA also inhibited TGF-ß2-induced phosphorylation of Akt and ERK, but did not inhibit Smad2/3 phosphorylation, the canonical pathway of TGF-ß signaling. Moreover, SAHA induced the expression of phosphatase and tensin homolog, a PI3K/Akt signaling factor, as well as bone morphogenetic protein 7, an endogenous antagonist of TGF-ß. These results imply that SAHA prevents TGF-ß2-induced increases in outflow resistance and regulates the non-Smad pathway of TGF-ß signaling in TM and MSC cells.

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.

An ex vivo model of human corneal rim perfusion organ culture.

The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 µl/min, the outflow facility was 0.359 ± 0.216 µl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.

In vivo measurement of trabecular meshwork stiffness in a corticosteroid-induced ocular hypertensive mouse model.

Ocular corticosteroids are commonly used clinically. Unfortunately, their administration frequently leads to ocular hypertension, i.e., elevated intraocular pressure (IOP), which, in turn, can progress to a form of glaucoma known as steroid-induced glaucoma. The pathophysiology of this condition is poorly understood yet shares similarities with the most common form of glaucoma. Using nanotechnology, we created a mouse model of corticosteroid-induced ocular hypertension. This model functionally and morphologically resembles human ocular hypertension, having titratable, robust, and sustained IOPs caused by increased resistance to aqueous humor outflow. Using this model, we then interrogated the biomechanical properties of the trabecular meshwork (TM), including the inner wall of Schlemm's canal (SC), tissues known to strongly influence IOP and to be altered in other forms of glaucoma. Specifically, using spectral domain optical coherence tomography, we observed that SC in corticosteroid-treated mice was more resistant to collapse at elevated IOPs, reflecting increased TM stiffness determined by inverse finite element modeling. Our noninvasive approach to monitoring TM stiffness in vivo is applicable to other forms of glaucoma and has significant potential to monitor TM function and thus positively affect the clinical care of glaucoma, the leading cause of irreversible blindness worldwide.

Nanoencapsulated hybrid compound SA-2 with long-lasting intraocular pressure-lowering activity in rodent eyes.

Purpose: Glaucoma is a neurodegenerative disease of the eye with an estimated prevalence of more than 111.8 million patients worldwide by 2040, with at least 6 to 8 million projected to become bilaterally blind. Clinically, the current method of slowing glaucomatous vision loss is to reduce intraocular pressure (IOP). In this manuscript, we describe the in vitro cytoprotective and in vivo long lasting IOP-lowering activity of the poly D, L-lactic-co-glycolic acid (PLGA) nanoparticle-encapsulated hybrid compound SA-2, possessing nitric oxide (NO) donating and superoxide radical scavenging functionalities. Methods: Previously characterized primary human trabecular meshwork (hTM) cells were used for the study. hTM cells were treated with SA-2 (100 µM, 200 µM, and 1,000 µM), SA-2 PLGA-loaded nanosuspension (SA-2 NPs, 0.1%), or vehicle for 30 min. Cyclic guanosine monophosphate (cGMP) and super oxide dismutase (SOD) levels were analyzed using commercial kits. In another experiment, hTM cells were pretreated with tert-butyl hydrogen peroxide (TBHP, 300 µM) for 30 min followed by treatment with escalating doses of SA-2 for 24 h, and CellTiter 96 cell proliferation assay was performed. For the biodistribution study, the cornea, aqueous humor, vitreous humor, retina, choroid, and sclera were collected after 1 h of administration of a single eye drop (30 µl) of SA-2 NPs (1% w/v) formulated in PBS to rat (n = 6) eyes. Compound SA-2 was quantified using high performance liquid chromatography /mass spectrometry (HPLC/MS). For the IOP-lowering activity study, a single SA-2 NPs (1%) eye drop was instilled in normotensive rats eyes and in the IOP-elevated rat eyes (n = 3/group, in the Morrison model of glaucoma), or Ad5TGFß2-induced ocular hypertensive (OHT) mouse eyes (n = 5/group). IOP was measured at various time points up to 72 h, and the experiment was repeated in triplicate. Mouse aqueous humor outflow facility was determined with multiple flow-rate infusion and episcleral venous pressure estimated with manometry. Results: SA-2 upregulated cGMP levels (six- to ten-fold) with an half maximal effective concentration (EC50) of 20.3 µM in the hTM cells and simultaneously upregulated (40-fold) the SOD enzyme when compared with the vehicle-treated hTM cells. SA-2 also protected hTM cells from TBHP-induced decrease in cell survival with an EC50 of 0.38 µM. A single dose of slow-release SA-2 NPs (1% w/v) delivered as an eye drop significantly lowered IOP (by 30%) in normotensive and OHT rodent eyes after 3 h post-dose, with the effect lasting up to 72 h. A statistically significant increase in aqueous outflow facility and a decrease in episcleral venous pressure was observed in rodents at this dose at 54 h. Conclusions: Hybrid compound SA-2 upregulated cGMP in hTM cells, increased outflow facility and decreased IOP in rodent models of OHT. Compound SA-2 possessing an antioxidant moiety provided additive cytoprotective activity to oxidatively stressed hTM cells by scavenging reactive oxygen species (ROS) and increasing SOD enzyme activity. Additionally, the PLGA nanosuspension formulation (SA-2 NPs) provided longer duration of IOP-lowering activity (up to 3 days) in comparison with the free non-encapsulated SA-2 drug. The data have implications for developing novel, non-prostaglandin therapeutics for IOP-lowering and cytoprotective effects with the possibility of an eye drop dosing regimen of once every 3 days for patients with glaucoma.

Effects of topical TGF-ß1, TGF-ß2, ATX, and LPA on IOP elevation and regulation of the conventional aqueous humor outflow pathway.

PURPOSE: The effects of aqueous mediators possibly increasing the outflow resistance, transforming growth factor-ß1 (TGF-ß1), TGF-ß2, autotaxin (ATX), and lysophosphatidic acid (LPA) on human trabecular meshwork (hTM) cells and monkey Schlemm's canal endothelial (SCE) cells were characterized and compared, and the effects of intracameral application of these mediators on intraocular (IOP) elevation were also examined. METHODS: Cells were treated with TGF-ß1, TGF-ß2, ATX, LPA, or vehicle, and mRNA and protein expression levels of α-SMA, COL1A1, fibronectin, ß-catenin, and ZO-1 were examined with real-time quantitative PCR (RT-qPCR) or immunofluorescence analyses or both. The permeability of cell monolayers was measured by determining the transendothelial electrical resistance (TEER) or with the fluorescein isothiocyanate (FITC)-dextran permeability assay. IOP was evaluated in rabbit eyes after intracameral administration of the mediators. RESULTS: All mediators induced upregulation of α-SMA, COL1A1, and fibronectin in hTM cells. The effect of TGF-ß2 on mRNA expression of fibrotic markers was statistically significantly greater than that of TGF-ß1. The effects of ATX and LPA indicated the time-dependent difference in the upregulation of α-SMA, COL1A1, and fibronectin. The TEER and FITC-dextran permeability of the SCE cells was evaluated after treatment with TGF-ß1 and TGF-ß2, but no statistically significant change was observed within 24 h. ATX and LPA also reduced permeability statistically significantly after 3 h and 0.5 h, respectively, and the effect of LPA was more rapid compared to that of ATX. Statistically significant IOP elevation was observed in rabbit eyes as early as 0.5-2.0 h after ATX and LPA treatment and at 24 h after treatment with TGF-ß2. CONCLUSIONS: TGF-ß2 and ATX and LPA regulate aqueous outflow by modulation of hTM cells and SCE cells, and differences in timing between the effects of each mediator were observed. ATX and LPA showed more rapid effects on IOP elevation than TGF-ß2. It was suggested that TGF-ß2 and ATX/LPA are involved in increases of IOP, but the timing and sustainability differ between mediators, and they may play specific roles in different glaucoma subtypes.

Circumferential trabecular meshwork cell density in the human eye.

The cells residing in the trabecular meshwork (TM) fulfill important roles in the maintenance of the tissue and the regulation of intraocular pressure (IOP). Here we examine (i) TM cell distribution along the circumference of the human eye, (ii) differences in TM cell density between regions of high and low outflow, and (iii) whether TM cell distribution in eyes from donors with primary open angle glaucoma (POAG) differs from that of normal eyes. Toward this end, the TM cell density from 12 radial segments around the circumference of the TM of human donor eyes (n = 6) with and without POAG was determined using histochemical methods. Areas of high, median, and low outflow were mapped in a different set of human donor eyes that were perfused in organ culture, and TM cell densities in these areas were determined in normal (n = 11) and POAG eyes (n = 6). Our analysis of 1380 tissue sections taken from the first set of six eyes shows that the average TM cell density of these six eyes ranges from 15.5 to 23.7 cells/100 µm and is negatively correlated to the maximum IOP recorded for each donor eye (R2 = 0.91). Considerable differences in TM cell density exist among sections taken from the same segment of an individual eye (average standard deviation = 2.35 cells/100 µm). Less variability is observed among the segment averages across the eye's circumference (average standard deviation = 1.03 cells/100 µm). Variations in cell density are similar between normal and POAG eyes and are not correlated with the anatomic position of examined segments (p = 0.745). The analysis of the second set of eyes shows that TM regions of high outflow display a TM cell density similar to regions of median or low outflow in both normal and POAG eyes. Together these findings demonstrate that (i) statistically significant differences in TM cell density exist along the circumference of each eye (ii) TM cellularity is not correlated with segmental flow and (iii) eyes with POAG, while displaying reduced TM cellularity, do not exhibit higher TM cell variability than normal eyes. Finally, statistical analysis of sections and segments indicates that measurements from 12 sections taken from 2 segments provide a reliable and cost-effective estimate of a human eye's TM cell density.

Adipose-derived stem cells integrate into trabecular meshwork with glaucoma treatment potential.

The trabecular meshwork (TM) is an ocular tissue that maintains intraocular pressure (IOP) within a physiologic range. Glaucoma patients have reduced TM cellularity and, frequently, elevated IOP. To establish a stem cell-based approach to restoring TM function and normalizing IOP, human adipose-derived stem cells (ADSCs) were induced to differentiate to TM cells in vitro. These ADSC-TM cells displayed a TM cell-like genotypic profile, became phagocytic, and responded to dexamethasone stimulation, characteristic of TM cells. After transplantation into naive mouse eyes, ADSCs and ADSC-TM cells integrated into the TM tissue, expressed TM cell markers, and maintained normal IOP, outflow facility, and extracellular matrix. Cell migration and affinity results indicated that the chemokine pair CXCR4/SDF1 may play an important role in ADSC-TM cell homing. Our study demonstrates the possibility of applying autologous or allogeneic ADSCs and ADSC-TM cells as a potential treatment to restore TM structure and function in glaucoma.

Identification of Estrogen Signaling in a Prioritization Study of Intraocular Pressure-Associated Genes.

Elevated intraocular pressure (IOP) is the only modifiable risk factor for primary open-angle glaucoma (POAG). Herein we sought to prioritize a set of previously identified IOP-associated genes using novel and previously published datasets. We identified several genes for future study, including several involved in cytoskeletal/extracellular matrix reorganization, cell adhesion, angiogenesis, and TGF-ß signaling. Our differential correlation analysis of IOP-associated genes identified 295 pairs of 201 genes with differential correlation. Pathway analysis identified ß-estradiol as the top upstream regulator of these genes with ESR1 mediating 25 interactions. Several genes (i.e., EFEMP1, FOXC1, and SPTBN1) regulated by ß-estradiol/ESR1 were highly expressed in non-glaucomatous human trabecular meshwork (TM) or Schlemm's canal (SC) cells and specifically expressed in TM/SC cell clusters defined by single-cell RNA-sequencing. We confirmed ESR1 gene and protein expression in human TM cells and TM/SC tissue with quantitative real-time PCR and immunofluorescence, respectively. 17ß-estradiol was identified in bovine, porcine, and human aqueous humor (AH) using ELISA. In conclusion, we have identified estrogen receptor signaling as a key modulator of several IOP-associated genes. The expression of ESR1 and these IOP-associated genes in TM/SC tissue and the presence of 17ß-estradiol in AH supports a role for estrogen signaling in IOP regulation.

The effects of negative periocular pressure on intraocular pressure.

Glaucoma is a major cause of blindness, and IOP reduction remains the only clinically-validated therapy. In this study, we analyze a novel IOP-lowering strategy that uses a modest negative pressure (vacuum) applied locally to the periorbital region by a pair of goggles with each lens individually connected to a programmable pump. Motivated by clinical data showing an IOP reduction, we used an existing validated lumped-parameter model of the eye to understand the putative mechanism of this treatment. The model considers aqueous humor dynamics, episcleral venous pressure, and changes in ocular blood volume to describe how IOP changes with time in response to an external perturbation. We find that clinical data are qualitatively and quantitatively consistent with model predictions if we include two primary mechanisms in the model: first, negative pressure application causes a relatively rapid increase in globe volume accompanied by increased blood volume in the eye. Second, negative pressure application reduces episcleral venous pressure, causing a slower adjustment of IOP due to altered aqueous humor dynamics. These results provide testable hypotheses that hopefully will lead to a fuller experimentally-driven understanding of how negative periocular pressure influences IOP. Evaluating the long-term effects of such treatments on glaucoma patients requires further clinical study.

Elevated pressure influences relative distribution of segmental regions of the trabecular meshwork.

Elevated intraocular pressure (IOP) is the primary risk factor for glaucoma and is the only treatable feature of the disease. There is a correlation between elevated pressure and homeostatic reductions in the aqueous humor outflow resistance via changes in the extracellular matrix of the trabecular meshwork. It is unclear how these extracellular matrix changes affect segmental patterns of aqueous humor outflow, nor do we understand their causal relationship. The goal of this study was to determine whether there are changes in the segmental outflow regions with perfusion in normal eyes, and whether these regions change during the IOP homeostatic response to elevated pressure. Using human anterior segment perfusion organ culture, we measured the amount of high flow (HF), intermediate flow (MF), and low flow (LF) regions before and after 7 days of perfusion at either physiologic pressure ("1x") or at elevated pressure ("2x"). We found a small but significant decrease in the amount of HF regions over 7 days perfusion at 1x pressure, and a twofold increase in the amount of MF regions over 7 days perfusion at 2x pressure. Small positional differences, or shifts in the specific location of HF, MF, or LF, occurred on a per eye basis and were not found to be statistically significant across biological replicates. Differences in the amount of segmental flow regions of contralateral eyes flowed at 1x pressure for 7 days were small and not statistically significant. These results demonstrate that perfusion at physiologic pressure had little effect on the distribution and amount of HF, MF and LF regions. However, the overall amount of MF regions is significantly increased in response to perfusion at elevated pressure during IOP homeostatic resistance adjustment. The amount of both HF and LF regions was decreased accordingly suggesting a coordinated response in the TM to elevated pressure.

Segmental differences found in aqueous angiographic-determined high - and low-flow regions of human trabecular meshwork.

This work sought to compare aqueous angiographic segmental patterns with bead-based methods which directly visualize segmental trabecular meshwork (TM) tracer trapping. Additionally, segmental protein expression differences between aqueous angiographic-derived low- and high-outflow human TM regions were evaluated. Post-mortem human eyes (One Legacy and San Diego eye banks; n = 15) were perfused with fluorescent tracers (fluorescein [2.5%], indocyanine green [0.4%], and/or fluorescent microspheres). After angiographic imaging (Spectralis HRA+OCT; Heidelberg Engineering), peri-limbal low- and high-angiographic flow regions were marked. Aqueous angiographic segmental outflow patterns were similar to fluorescent microsphere TM trapping segmental patterns. TM was dissected from low- and high-flow areas and processed for immunofluorescence or Western blot and compared. Versican expression was relatively elevated in low-flow regions while MMP3 and collagen VI were relatively elevated in high-flow regions. TGF-ß2, thrombospondin-1, TGF-ß receptor1, and TGF-ß downstream proteins such as α-smooth muscle actin were relatively elevated in low-flow regions. Additionally, fibronectin (FN) levels were unchanged, but the EDA isoform (FN-EDA) that is associated with fibrosis was relatively elevated in low-flow regions. These results show that segmental aqueous angiographic patterns are reflective of underlying TM molecular characteristics and demonstrate increased pro-fibrotic activation in low-flow regions. Thus, we provide evidence that aqueous angiography outflow visualization, the only tracer outflow imaging method available to clinicians, is in part representative of TM biology.

Functional, structural, and molecular identification of lymphatic outflow from subconjunctival blebs.

The purpose of this study is to evaluate outflow pathways from subconjunctival blebs and to identify their identity. Post-mortem porcine (n = 20), human (n = 1), and bovine (n = 1) eyes were acquired, and tracers (fluorescein, indocyanine green, or fixable/fluorescent dextrans) were injected into the subconjunctival space to create raised blebs where outflow pathways were visualized qualitatively and quantitatively. Rodents with fluorescent reporter transgenes were imaged for structural comparison. Concurrent optical coherence tomography (OCT) was obtained to study the structural nature of these pathways. Using fixable/fluorescent dextrans, tracers were trapped to the bleb outflow pathway lumen walls for histological visualization and molecular identification using immunofluorescence against lymphatic and blood vessel markers. Bleb outflow pathways could be observed using all tracers in all species. Quantitative analysis showed that the nasal quadrant had more bleb-related outflow pathways compared to the temporal quadrant (nasal: 1.9±0.3 pathways vs. temporal: 0.7±0.2 pathways; p = 0.003). However, not all blebs resulted in an outflow pathway (0-pathways = 18.2%; 1-pathway = 36.4%; 2-pathways = 38.6%; and 3-pathways = 6.8%). Outflow signal was validated as true luminal pathways using optical coherence tomography and histology. Bicuspid valves were identified in the direction of flow in porcine eyes. Immunofluorescence of labeled pathways demonstrated a lymphatic (Prox-1 and podoplanin) but not a blood vessel (CD31) identity. Therefore, subconjunctival bleb outflow occurs in discrete luminal pathways. They are lymphatic as assessed by structural identification of valves and molecular identification of lymphatic markers. Better understanding of lymphatic outflow may lead to improved eye care for glaucoma surgery and ocular drug delivery.

Circumferential canal surgery: a brief history.

PURPOSE OF REVIEW: Most microinvasive glaucoma surgery (MIGS) procedures bypass outflow resistance residing proximally in the trabecular meshwork and inner wall of Schlemm's canal. A novel procedure combining trabeculotomy with viscodilation adds to this by also addressing distal resistance of the canal and collector channel ostia. This review examines the development and evidence for both trabeculotomy and canaloplasty separately and the combination in a single procedure. RECENT FINDINGS: Recent aqueous angiography studies have confirmed the segmental nature of outflow through Schlemm's canal highlighting the need to address distal outflow pathway resistance. Combined trabeculotomy and viscodilation ab interno is a novel approach with a new purpose-designed device (OMNI Surgical System) becoming available to surgeons in early 2018. Recent results as both a standalone and combined with cataract procedure demonstrate significant intraocular pressure reductions with an average 41% reduction from baseline in the pseudophakic group. SUMMARY: Targeting both distal as well as proximal points of outflow resistance in the conventional pathway may prove to be a highly efficacious MIGS modality. Additional large prospective studies are currently ongoing to confirm these preliminary results.

Dose-dependent effects of netarsudil, a Rho-kinase inhibitor, on the distal outflow tract.

PURPOSE: To characterize the effects of netarsudil on the aqueous humor outflow tract distal to the trabecular meshwork (TM). We hypothesized that netarsudil increases outflow facility in eyes with and without circumferential ab interno trabeculectomy (AIT) that removes the TM. METHODS: Sixty-four porcine anterior segment cultures were randomly assigned to groups with (n = 32) and without circumferential AIT (n = 32). Cultures were exposed to 0.1, 1, and 10 µM netarsudil (N = 8 eyes per concentration). For each concentration, IOP and vessel diameters were compared with their respective pretreatment baselines. Outflow tract vessel diameters were assessed by spectral-domain optical coherence tomography (SDOCT) and rendered in 4D (XYZ time series). RESULTS: Netarsudil at 1 µM reduced IOP both in eyes with TM (- 0.60 ± 0.24 mmHg, p = 0.01) and in eyes without TM (- 1.79 ± 0.42 mmHg, p < 0.01). At this concentration, vessels of the distal outflow tract dilated by 72%. However, at 0.1 µM netarsudil elevated IOP in eyes with TM (1.59 ± 0.36 mmHg, p < 0.001) as well as in eyes without TM (0.23 ± 0.32 mmHg, p < 0.001). Vessels of the distal outflow tract constricted by 31%. Similarly, netarsudil at a concentration of 10 µM elevated IOP both in eyes with TM (1.91 ± 0.193, p < 0.001) and in eyes without TM (3.65 ± 0.86 mmHg, p < 0.001). At this concentration, outflow tract vessels constricted by 27%. CONCLUSION: In the porcine anterior segment culture, the dose-dependent IOP changes caused by netarsudil matched the diameter changes of distal outflow tract vessels. Hyper- and hypotensive properties of netarsudil persisted after TM removal.