Outflow Facility Research Articles

Ultrasound-activated perfluorocarbon nanodroplets in ophthalmology

Glaucoma, a leading cause of blindness, can result from increased intraocular pressure resulting from elevated outflow resistance. In this preliminary study, we consider the potential of ultrasound-activated perfluorocarbon nanodroplets (NDs) to increase the permeability of the outflow pathway. 100nm diameter NDs with a perfluoropentane core and lipid shell were introduced into the anterior chamber (AC) of ex vivo pig and in vivo rat eyes. Imaging (18 and 28 MHz) was performed with a weakly focused (F = 2.9) beam. Activation was induced by increasing the array sub-aperture to tighten the focus to F = 0.62 centrally. Images showed NDs to be distributed widely within the AC, from which they could enter the outflow pathway by virtue of their small dimension. At 28 MHz, NDs were activated at a peak negative pressure of 5 MPa, corresponding to a mechanical index of 1.8.Two days after treatment, rat eyes showed no sign of inflammation, but a few small gas bubbles were present in the AC. In this application, NDs are advantageous as cavitation nuclei with respect to conventional microbubble contrast agents because of their higher density and smaller diameter (<1/10th that of microbubble agents), which facilitate entry into the outflow channels. Future preclinical in vivo studies will assess the effect of treatment on IOP and outflow facility.

MiR-18a-5p Targets Connective Tissue Growth Factor Expression and Inhibits Transforming Growth Factor β2-Induced Trabecular Meshwork Cell Contractility.

Increased trabecular meshwork (TM) cell and tissue contractility is a driver of the reduced outflow facility and elevation of intraocular pressure (IOP) associated with primary open-angle glaucoma (POAG). Connective tissue growth factor (CTGF) is an established mediator of TM cell contractility, and its expression is increased in POAG due to transforming growth factor β 2 (TGFβ2) signalling. Inhibiting CTGF upregulation using microRNA (miRNA) mimetics could represent a new treatment option for POAG. A combination of in silico predictive tools and a literature review identified a panel of putative CTGF-targeting miRNAs. Treatment of primary human TM cells with 5 ng/mL TGFβ2 for 24 h identified miR-18a-5p as a consistent responder, being upregulated in cells from five different human donors. Transfection of primary donor TM cells with 20 nM synthetic miR-18a-5p mimic reduced TGFβ2-induced CTGF protein expression, and stable lentiviral-mediated overexpression of this miRNA reduced TGFβ2-induced contraction of collagen gels. Together, these findings identify miR-18a-5p as a mediator of the TGFβ2 response and a candidate therapeutic agent for glaucoma via its ability to inhibit CTGF-associated increased TM contractility.

Endothelial Glycocalyx Morphology in Different Flow Regions of the Aqueous Outflow Pathway of Normal and Laser-Induced Glaucoma Monkey Eyes.

Glycocalyx morphology was examined in the trabecular outflow pathway of monkey eyes with and without experimental glaucoma. Laser burns were administered along ~270 degrees of the trabecular meshwork (TM) of one eye (n = 6) or both eyes (n = 2) of each monkey until intraocular pressure remained elevated. Portions of the TM were not laser-treated. Unlasered eyes (n = 6) served as controls. Enucleated eyes were perfused at 15 mmHg to measure the outflow facility, perfused with fluorescein to evaluate the outflow pattern, perfusion-fixed for glycocalyx labeling, and processed for electron microscopy. Coverage and thickness of the glycocalyx were measured in the TM, Schlemm’s canal (SC), collector channels (CCs), intrascleral veins (ISVs), and episcleral veins (ESVs) in non-lasered regions and high- and low-flow regions of controls. Compared to controls, laser-treated eyes had decreased outflow facility (p = 0.02). Glycocalyx thickness increased from the TM to ESVs in non-lasered regions and controls (p < 0.05). Glycocalyx coverage was generally greater distally in non-lasered regions (p < 0.05). In lasered regions, TM, SC, and CCs were partly to completely obliterated, and ISVs and ESVs displayed minimal glycocalyx. Whether the glycocalyx is decreased in the trabecular outflow pathway of human glaucomatous eyes warrants 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.

A Novel Mouse Model of TGFβ2-Induced Ocular Hypertension Using Lentiviral Gene Delivery.

Glaucoma is a multifactorial disease leading to irreversible blindness. Primary open-angle glaucoma (POAG) is the most common form and is associated with the elevation of intraocular pressure (IOP). Reduced aqueous humor (AH) outflow due to trabecular meshwork (TM) dysfunction is responsible for IOP elevation in POAG. Extracellular matrix (ECM) accumulation, actin cytoskeletal reorganization, and stiffening of the TM are associated with increased outflow resistance. Transforming growth factor (TGF) β2, a profibrotic cytokine, is known to play an important role in the development of ocular hypertension (OHT) in POAG. An appropriate mouse model is critical in understanding the underlying molecular mechanism of TGFβ2-induced OHT. To achieve this, TM can be targeted with recombinant viral vectors to express a gene of interest. Lentiviruses (LV) are known for their tropism towards TM with stable transgene expression and low immunogenicity. We, therefore, developed a novel mouse model of IOP elevation using LV gene transfer of active human TGFβ2 in the TM. We developed an LV vector-encoding active hTGFβ2C226,228S under the control of a cytomegalovirus (CMV) promoter. Adult C57BL/6J mice were injected intravitreally with LV expressing null or hTGFβ2C226,228S. We observed a significant increase in IOP 3 weeks post-injection compared to control eyes with an average delta change of 3.3 mmHg. IOP stayed elevated up to 7 weeks post-injection, which correlated with a significant drop in the AH outflow facility (40.36%). Increased expression of active TGFβ2 was observed in both AH and anterior segment samples of injected mice. The morphological assessment of the mouse TM region via hematoxylin and eosin (H&E) staining and direct ophthalmoscopy examination revealed no visible signs of inflammation or other ocular abnormalities in the injected eyes. Furthermore, transduction of primary human TM cells with LV_hTGFβ2C226,228S exhibited alterations in actin cytoskeleton structures, including the formation of F-actin stress fibers and crossed-linked actin networks (CLANs), which are signature arrangements of actin cytoskeleton observed in the stiffer fibrotic-like TM. Our study demonstrated a mouse model of sustained IOP elevation via lentiviral gene delivery of active hTGFβ2C226,228S that induces TM dysfunction and outflow resistance.

Potential Mechanisms of Intraocular Pressure Reduction by Micropulse Transscleral Cyclophotocoagulation in Rabbit Eyes.

PurposeTo evaluate the histological changes associated with, and the potential mechanisms of, intraocular pressure (IOP) reduction by micropulse cyclophotocoagulation (MP-CPC) in rabbit eyes.MethodsMP-CPC was performed on the right eyes of Dutch belted rabbits, whereas the left eyes served as controls. The laser power settings were 250, 500, 750, 1000, 1500, and 2000 mW, 10 seconds per sweep, 100 seconds in total. IOP, outflow facility, and uveoscleral outflow tract imaging, using a fluorescent tracer, were examined at one week after MP-CPC. Changes of morphology and protein expressions in the outflow tissues, conjunctiva, and sclera were also evaluated.ResultsSignificant reductions in IOP after MP-CPC were observed at 500 to 1000 mW (P = 0.036 and P = 0.014, respectively). The pre-MP-CPC IOP was 11.35 ± 0.41 mm Hg. At one week after surgery, the respective IOP values in the eyes treated at 500 mW and 1000 mW were 9.45 ± 0.49 mm Hg and 7.4 ± 0.27 mm Hg, respectively. Severe ciliary body damage was observed at 1500 to 2000 mW. MMP1–3 and fibronectin expression levels in the outflow tract and ciliary body were upregulated after MP-CPC. The α-smooth muscle actin (α-SMA) was upregulated at higher power levels. MP-CPC significantly increased uveoscleral outflow, whereas the outflow facility did not change. The α-SMA, collagen, and fibronectin were significantly upregulated in the subconjunctiva and sclera.ConclusionsReactive fibrotic responses were observed in the outflow tract, conjunctiva, and sclera after MP-CPC. A potential mechanism of IOP reduction by MP-CPC in pigmented rabbit eyes may involve increased uveoscleral outflow related to MMP upregulation.

Transgene expression of Stanniocalcin-1 provides sustained intraocular pressure reduction by increasing outflow facility.

Glaucoma is the leading cause of irreversible blindness worldwide. Therapies for glaucoma are directed toward reducing intraocular pressure (IOP), the leading risk factor and only reliable therapeutic target via topical medications or with procedural intervention including laser or surgery. Though topical therapeutics are typically first line, less than 50% of patients take drops as prescribed. Sustained release technologies that decrease IOP for extended periods of time are being examined for clinical use. We recently identified Stanniocalcin-1, a naturally occurring hormone, as an IOP-lowering agent. Here, we show that a single injection into the anterior chamber of mice with an adeno-associated viral vector containing the transgene of stanniocalcin-1 results in diffuse and sustained expression of the protein and produces IOP reduction for up to 6 months. As the treatment effect begins to wane, IOP-lowering can be rescued with a repeat injection. Aqueous humor dynamic studies revealed an increase in outflow facility as the mechanism of action. This first-in-class therapeutic approach has the potential to improve care and reduce the rates of vision loss in the 80 million people worldwide currently affected by glaucoma.

The hypotensive effect of dosed endotrabeculoectomy in combination with non-penetrating deep sclerectomy in patients with primary open-angle glaucoma

Aim. To analyze the effect of dosed endotrabeculectomy in combination with non-penetrating deep sclerectomy on the level of intraocular pressure (IOP) and aqueous humor outflow from the anterior chamber. Materials and methods. The study involved 12 patients (12 eyes) who underwent dosed endotrabeculectomy with ab interno approach in combination with non-penetrating deep sclerectomy surgery. All the patients had moderate or severe trabecular pigmentation by gonioscopy. The follow-up period was 12 months. Measurement of IOP was performed according to Maklakov before and after surgery (on the 7th day, 1st, 3rd, 6th and 12th month). Tonography was performed according to Nesterov (using the simplified method) at the same time periods. The outflow facility coefficient and Becker’s coefficient were determined. The number of local antihypertensive agents used within that period was also observed. A combined drug was counted as two – according to the number of active substances. Results. The difference between preoperative and postoperative IOP was found to be significant up to and including the 12th month. IOP at the 6th month after surgery was 8.67 mm Hg lower and at the 12th month – 7.83 mm Hg lower. The outflow facility coefficient was significantly increased from 0.14 ± 0.06 mm3/mm Hg/min up to 0.32 ± 0.03 mm3/mm Hg/min (P &lt; 0.05). Becker’s coefficient was significantly decreased from 157.80 ± 8.04 to 42.2 ± 3.6 (P &lt; 0.05). Conclusions. Dosed endotrabeculoectomy in combination with non-penetrating deep sclerectomy has shown the significant hypotensive effect, as well as the increase in outflow facility coefficient and the decrease in Becker’s coefficient during the postoperative period among patients with primary open-angle glaucoma within the 12-month follow-up.

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.

Trabecular Meshwork Cholesterol Levels Regulate Actin Polymerization and Tunneling Nanotubes

Trabecular meshwork (TM) tissue is made of highly contractile mechanosensitive cells regulating aqueous humor (AH) outflow facility and intraocular pressure (IOP). Increased actin‐mediated contraction of TM elevates IOP causing glaucoma and irreversible blindness. This study aims at understanding the role of cholesterol in regulating actin dynamics in human TM (HTM) cells. To test this, HTM cells were treated with SiR‐Actin in serum‐free media overnight and F‐actin changes were recorded using live‐cell time‐lapse (5 min interval) confocal imaging before and during exposure to ‐ A) 10mM methyl‐β‐cyclodextrin (MβCD) for 1h for acute removal of cellular cholesterol, B) 100μM cholesterol‐saturated MβCD (MβCD‐CHOL) for 1h to enrich cholesterol, and C) 10mM MβCD for 1h followed by cholesterol supplementation using MβCD‐CHOL for 1h. F‐actin fluorescence intensity changes were calculated and unpaired t‐test was used for statistical analyses and results were significant if p&lt;0.05 with a sample size of n= 4‐57. HTM cells plated on coverslips were treated as A – C as mentioned above, cholesterol and lipid synthesis inhibitors for 24h ‐ D) 100μM atorvastatin and E) 20μM fatostatin, respectively, F) actin polymerization disruptor ‐ 10μM cytochalasin D (CYTD) for 1h, G) 10μM CYTD for 1h followed by 100μM MβCD‐CHOL for 1h, H) microtubule disruptor ‐ 1μM demecolcine (DEM) for 1h, I) 1μM DEM for 1h followed by 100μM MβCD‐CHOL for 1h. Cells without treatment were used as control. After all these treatments, cells were fixed and immunofluorescence (IF) was performed to check for cholesterol using filipin, F‐actin by phalloidin, focal adhesion (FA) localization based on ‐ vinculin and paxillin distribution. Further F‐actin/G‐actin in vitro assay was performed to quantify the changes in actin polymerization under the conditions A – I compared to the control. The imaging analysis showed cholesterol removal significantly decreased F‐actin fluorescence intensity from 20min sustaining up to 1h and decreased the FA distribution. Cholesterol supplementation led to partial rescue of actin and FA indicating the re‐establishment of actin cytoskeleton and increased tunneling nanotube structures enhancing the cell‐cell communication (Figure 1). Moreover, IF results show that atorvastatin and fatostatin decreased F‐actin and FA distribution. CYTD disrupted F‐actin network leaving remnant filament ends and decreased FA but cholesterol enrichment increased F‐actin branches formed at ~70‐degree angle indicating Arp2/3 activation. DEM treatment increased F‐actin and FA, and cholesterol enrichment induced thick F‐actin filaments. These results were confirmed by quantitative F/G‐actin ratio decrease and increase upon cholesterol removal and enrichment, respectively. Additionally, supplementing cholesterol after removal increased the ratio compared to solely removing cholesterol. Interestingly, F/G‐actin ratio increased under CYTD treatment indicating that CYTD disrupted longer actin filaments but helped polymerization of newly formed actin filaments. Finally, DEM treatment increased F/G‐actin ratio, which was further increased by cholesterol enrichment. This systematic evaluation of cholesterol modulation on TM actin cytoskeleton polymerization identifies the significance of maintaining membrane and cellular cholesterol levels and cellular communication for achieving IOP homeostasis.

Efficacy and Patient Tolerability of Omidenepag Isopropyl in the Treatment of Glaucoma and Ocular Hypertension.

Current therapeutic approaches for glaucoma aim to reduce intraocular pressure (IOP), which is the only available and reliable strategy proven to control the risk of disease development and progression. Omidenepag isopropyl (OMDI) is a novel topical ocular hypotensive agent that was launched onto the market for the treatment of glaucoma and ocular hypertension (OHT). After topical instillation and during corneal penetration, OMDI is converted into the active metabolite omidenepag (OMD), which behaves as a non-prostaglandin, selective E-prostanoid subtype 2 (EP2) receptor agonist. The topical administration of 0.002% OMDI once-daily (QD) possesses a 20–35% IOP-lowering effect, comparable to that of prostaglandin analogs targeting F-prostanoid (FP) receptor QD, which are the current first-line for pharmaceutical reduction of IOP. However, the mechanism of action and adverse events (AEs) of OMDI are different from those of FP receptor agonists. OMDI reduces IOP by enhancing both conventional trabecular and uveoscleral outflow facilities without complications of prostaglandin-associated periorbitopathy (PAP) seen with FP receptor agonists. Moreover, OMDI was also effective and well-tolerated in non-/poor responders to latanoprost and showed a stable IOP-lowering effect for one year, and its concomitant use with timolol enhanced the IOP-lowering effect. OMDI demonstrated acceptable safety and tolerability with good adherence and can be used in almost every patient. However, OMDI has some AEs such as conjunctival hyperemia, corneal thickening, macular edema/cystoid macular edema and ocular inflammation. Moreover, OMDI is contraindicated in patients who are allergic to the product, in aphakic or pseudophakic eyes, and in combination with tafluprost eye drops. If used appropriately in the right patients, OMDI could be an effective treatment option for glaucoma and OHT as a first-line alternative to FP agonists. Here, we summarize the results of clinical studies of OMDI and discuss its efficacy and patient tolerability in glaucoma and OHT in this review.

Emergent Temporal Signaling in Human Trabecular Meshwork Cells: Role of TRPV4-TRPM4 Interactions.

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.

A Role of Caveolae in Trabecular Meshwork Mechanosensing and Contractile Tone.

Polymorphisms in the CAV1/2 gene loci impart increased risk for primary open-angle glaucoma (POAG). CAV1 encodes caveolin-1 (Cav1), which is required for biosynthesis of plasma membrane invaginations called caveolae. Cav1 knockout mice exhibit elevated intraocular pressure (IOP) and decreased outflow facility, but the mechanistic role of Cav1 in IOP homeostasis is unknown. We hypothesized that caveolae sequester/inhibit RhoA, to regulate trabecular meshwork (TM) mechanosensing and contractile tone. Using phosphorylated myosin light chain (pMLC) as a surrogate indicator for Rho/ROCK activity and contractile tone, we found that pMLC was elevated in Cav1-deficient TM cells compared to control (131 ± 10%, n = 10, p = 0.016). Elevation of pMLC levels following Cav1 knockdown occurred in cells on a soft surface (137 ± 7%, n = 24, p < 0.0001), but not on a hard surface (122 ± 17%, n = 12, p = 0.22). In Cav1-deficient TM cells where pMLC was elevated, Rho activity was also increased (123 ± 7%, n = 6, p = 0.017), suggesting activation of the Rho/ROCK pathway. Cyclic stretch reduced pMLC/MLC levels in TM cells (69 ± 7% n = 9, p = 0.002) and in Cav1-deficient TM cells, although not significantly (77 ± 11% n = 10, p = 0.059). Treatment with the Cav1 scaffolding domain mimetic, cavtratin (1 μM) caused a reduction in pMLC (70 ± 5% n = 7, p = 0.001), as did treatment with the scaffolding domain mutant cavnoxin (1 μM) (82 ± 7% n = 7, p = 0.04). Data suggest that caveolae differentially regulate RhoA signaling, and that caveolae participate in TM mechanotransduction. Cav1 regulation of these key TM functions provide evidence for underlying mechanisms linking polymorphisms in the Cav1/2 gene loci with increased POAG risk.

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.

Early-Onset Glaucoma in egl1 Mice Homozygous for Pitx2 Mutation.

Mutations in PITX2 cause Axenfeld–Rieger syndrome, with congenital glaucoma as an ocular feature. The egl1 mouse strain carries a chemically induced Pitx2 mutation and develops early-onset glaucoma. In this study, we characterized the glaucomatous features in egl1 mice. The eyes of egl1 and C57BL/6J control mice were assessed by slit lamp examination, total aqueous humor outflow facility, intraocular pressure (IOP) measurement, pattern electroretinography (PERG) recording, and histologic and immunohistochemistry assessment beginning at 3 weeks and up to 12 months of age. The egl1 mice developed elevated IOP as early as 4 weeks old. The IOP elevation was variable and asymmetric within and between the animals. The aqueous humor outflow facility was significantly reduced in 12-month-old animals. PERG detected a decreased response at 2 weeks after the development of IOP elevation. Retinal ganglion cell (RGC) loss was detected after 8 weeks of IOP elevation. Slit lamp and histologic evaluation revealed corneal opacity, iridocorneal adhesions (anterior synechiae), and ciliary body atrophy in egl1 mice. Immunohistochemistry assessment demonstrated glial cell activation and RGC axonal injury in response to IOP elevation. These results show that the eyes of egl1 mice exhibit anterior segment dysgenesis and early-onset glaucoma. The egl1 mouse strain may represent a useful model for the study of congenital glaucoma.

Anterior Segment Anatomy and Conventional Outflow Physiology of the Tree Shrew (Tupaia belangeri).

PurposeRodent 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.MethodsAqueous 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.ResultsAnterior 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).ConclusionsThese 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.

Severe Intraocular Pressure Fluctuation following Hemodialysis after Cataract Surgery in a Patient with Chronic Renal Failure

Purpose: To report a case of severe intraocular pressure fluctuation following hemodialysis after cataract surgery in a patient with chronic renal failure.Case summary: A 51-year-old female patient on hemodialysis for chronic renal failure, who was seen regularly by our retina clinic for proliferative diabetic retinopathy and right eye cataract, presented with decreased visual acuity in her right eye. During phacoemulsification under the diagnosis of right eye cataract, zonular dialysis and posterior capsule rupture occurred and intraocular lens ciliary sulcus insertion was performed. Following hemodialysis 3 days after the surgery, the patient complained of headache and right eye pain. The intraocular pressure of the right eye had increased to 44 mmHg. Mild inflammation in the anterior chamber was found with remnant lens material present at the inferior anterior angle. The use of glaucoma eye drops, anterior chamber paracentesis, and anterior chamber irrigation reduced the intraocular pressure and improved the patient’s symptoms. However, a severe increase in intraocular pressure recurred with the above symptoms, again following hemodialysis. Intraocular pressure fluctuation was repetitively present after serial hemodialysis treatments for 1 month, at which point cataract and glaucoma surgeries were considered. However, the intraocular pressure stabilized afterwards; thus, the patient remained under observation and was treated with glaucoma eye drops.Conclusions: Inflammation and a decrease in the aqueous outflow facility due to remnant lens material and the use of an ophthalmic viscoelastic device in cataract surgery may cause severe intraocular pressure fluctuations in patients undergoing hemodialysis for chronic renal failure.

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.

Effects of selective EP2 receptor agonist, omidenepag, on trabecular meshwork cells, Schlemm\u2019s canal endothelial cells and ciliary muscle contraction

This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm’s canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.

Selective Laser Trabeculoplasty in the Treatment of Ocular Hypertension and Open-Angle Glaucoma: Clinical Review.

Selective laser trabeculoplasty (SLT) is a glaucoma treatment that reduces intraocular pressure (IOP). Its mechanism is based on the biological effects of the selective application of laser energy to pigmented trabecular meshwork (TM) cells, resulting in increased outflow facility. Herein, we review current publications on SLT and summarize its efficacy and safety for different indications in open-angle glaucoma (OAG) and ocular hypertension (OHT) treatment. SLT effectively reduces IOP when used as a primary treatment. In patients whose IOP is medically controlled, SLT helps to reduce medication use, and when maximally tolerated topical therapy is ineffective, SLT facilitates the realization of the target IOP. SLT is a repeatable procedure for which the vast majority of complications are mild and self-limiting. With effective IOP reduction, low complication rates and the potential to repeat the procedure, SLT offers the possibility of delaying the introduction of medical therapy and other more invasive treatment modalities while simultaneously avoiding the accompanying complications. With this knowledge, we suggest that SLT be considered as an essential primary treatment option in OAG and OHT, switching to other treatment modalities only when laser procedures are insufficient for achieving the required target IOP.