Trabecular Pathway Research Articles

Protocol for the Extraction and Characterization of Trabecular Meshwork Cell Cytoskeleton Fraction.

Among various cellular attributes modulating aqueous humor (AH) outflow through the trabecular pathway and eventually intraocular pressure, the involvement of actomyosin regulated cellular contraction and relaxation, cell-extracellular matrix adhesion and cell-cell junctions, and mechanotransduction are well recognized. Although various biological and pharmacological agent-modulated AH outflows were associated with altered actin cytoskeletal organization and cell adhesive interactions of trabecular meshwork (TM) cells, these changes were analyzed largely with a biased approach to examine the specific proteins, but there were very few efforts in examining them with hypothesis-free unbiased approach in their native state. Therefore, in this chapter, we describe a protocol tailored to characterize the cytoskeleton of TM cells. This simple protocol can be applied to identifying the differentially regulated TM cell cytoskeleton proteins under any given treatment condition in conjunction with quantitative proteomic analysis.

Netarsudil monotherapy as the initial treatment for open-angle glaucoma and ocular hypertension in Indian patients: A real-world evaluation of efficacy and safety.

Glaucoma is the second leading cause of blindness worldwide, affecting more than 64 million people aged 40-80. The best way to manage primary open-angle glaucoma (POAG) is by lowering the intraocular pressure (IOP). Netarsudil is a Rho kinase inhibitor, the only class of antiglaucoma medications that reorganizes the extracellular matrix to improve the aqueous outflow through the trabecular pathway. An open-label, real-world, multicentric, observation-based 3-month study was performed for assessing the safety and ocular hypotensive efficacy of netarsudil ophthalmic solution (0.02% w/v) in patients with elevated IOP. Patients were given netarsudil ophthalmic solution (0.02% w/v) as a first-line therapy. Diurnal IOP measurements, best-corrected visual acuity, and adverse event assessments were recorded at each of the five visits (Day-1: screening day and first dosing day; subsequent observations were taken at 2 weeks, 4 weeks, 6 weeks, and 3 months). Four hundred and sixty-nine patients from 39 centers throughout India completed the study. The mean IOP at baseline of the affected eyes was 24.84 ± 6.39 mmHg (mean ± standard deviation). After the first dose, the IOP was measured after 2, 4, and 6 weeks, with the final measurement taken at 3 months. The percentage reduction in IOP in glaucoma patients after 3 months of once-daily netarsudil 0.02% w/v solution use was 33.34%. The adverse effects experienced by patients were not severe in the majority of cases. Some adverse effects observed were redness, irritation, itching, and others, but only a small number of patients experienced severe reactions, as reported in a decreasing order: redness > irritation > watering > itching > stinging > blurring. We found that netarsudil 0.02% w/v solution monotherapy when used as the first-line treatment in primary open-angle glaucoma and ocular hypertension was both safe and effective.

Netarsudil for the Treatment of Open-Angle Glaucoma and Ocular Hypertension: A Literature Review.

To evaluate netarsudil's role as first-line therapy for the treatment of open-angle glaucoma (OAG) and ocular hypertension (OHT). A literature search utilizing MEDLINE and CINAHL was performed using netarsudil and AR-13324 as keywords. Studies published from January 1970 to September 2020 were eligible. For inclusion, articles were required to be published in English and participants enrolled in phase I, II, or III clinical trials. Articles were excluded if netarsudil was coformulated with another medication. Preclinical research, case reports, case series, review articles, citations without an abstract, and newsletters were excluded. The search retrieved 97 unique citations; 90 results were excluded, and 7 studies were included for analysis. In all, 20 years elapsed between the Food and Drug Administration's approvals of distinct medications to treat OAG. Existing first-line therapies target the uveoscleral pathway, which is responsible for a small amount of aqueous humor outflow. Rho kinase inhibitors target the trabecular pathway, which is responsible for 90% of aqueous humor outflow; thus, Rho kinase inhibitors may significantly reduce intraocular pressure and improve clinical outcomes for patients with OAG or OHT. Evidence demonstrates that netarsudil is inferior to prostaglandin analogues and noninferior to topical β-blockers in the treatment of OAG and OHT. Hyperemia is a common adverse drug reaction, which often resolves after medication discontinuation. Additional phase III clinical trials and evidence-based guidelines are necessary to determine netarsudil's position in OAG and OHT management.

New glaucoma medications: latanoprostene bunod, netarsudil, and fixed combination netarsudil-latanoprost.

Reduction of intraocular pressure is the only proven method to treat glaucoma. Initial treatment of glaucoma commonly involves using anti-glaucoma medications either as monotherapy or combination therapy. Studies on aqueous humour dynamics have contributed to our understanding of aqueous outflow mechanisms that have led to the discovery of new drugs. Three new drugs (latanoprostene bunod 0.24%, netarsudil 0.02%, and fixed combination netarsudil 0.02% -latanoprost 0.005%) have been introduced recently in the market with novel mechanisms of action. Latanoprostene bunod 0.024% is a nitric oxide-donating prostaglandin F2α analogue which increases the aqueous outflow both by uveoscleral and trabecular pathways. Netarsudil 0.02% is a potent Rho kinase/norepinephrine transporter inhibitor acting by increasing the trabecular outflow, decreasing the aqueous production, and possibly decreasing the episcleral venous pressure. This review highlights the role of these drugs in the management of glaucoma, with an overview of the major clinical trials on their efficacy, safety, and tolerability.

Development of a Novel Intraocular-Pressure-Lowering Therapy Targeting ATX.

Elevated intraocular pressure (IOP) is the major cause of glaucoma, which is the second leading cause of blindness. However, current glaucoma treatments cannot completely regulate IOP and progression of glaucoma. Our group recently found that autotaxin (ATX) activity in human aqueous humor (AH) was positively correlated with increased IOP in various subtypes of glaucoma. To develop new IOP-lowering treatments, we generated a novel ATX inhibitor as an ophthalmic drug by high-throughput screening, followed by inhibitor optimization. Administration of the optimized ATX inhibitor (Aiprenon) reduced IOP in laser-treated mice exhibiting elevated IOP and higher level of ATX activity in AH and normal mice in vivo. The stimulation of ATX induced outflow resistance in the trabecular pathway; however, administration of Aiprenon recovered the outflow resistance in vitro. The in vitro experiments implied that the IOP-lowering effect of Aiprenon could be correlated with the altered cellular behavior of trabecular meshwork (TM) and Schlemm's canal endothelial (SC) cells. Overall, our findings showed that ATX had major impact in regulating IOP as a target molecule, and potent ATX inhibitors such as Aiprenon could be a promising therapeutic approach for lowering IOP.

Netarsudil/latanoprost fixed-dose combination for the treatment of open-angle glaucoma or ocular hypertension.

The fixed-dose combination (FDC) of netarsudil 0.02%/ latanoprost 0.005% was approved by the United States Food and Drug Administration (FDA) on March 12, 2019, for the reduction of intraocular pressure (IOP) in patients with open-angle glaucoma (OAG) and ocular hypertension (OHT). Netarsudil is a Rho kinase (ROCK) inhibitor and latanoprost is a prostaglandin analogue (PGA). Once-daily administration of this FDC reduces IOP by enhancing aqueous outflow through both the trabecular pathways (ROCK inhibition) and uveoscleral pathways (PGA). Two phase III clinical trials, MERCURY-1 and MERCURY-2, confirmed significantly greater efficacy of the FDC than the individual components, with IOP reductions of 30% or greater observed in 59-65% of subjects treated with FDC compared with 29-37% of subjects treated with latanoprost alone and 21-29% of subjects treated with netarsudil alone. The FDC was well tolerated with mostly mild ocular side effects and limited systemic side effects. This paper will review the work leading to FDA approval and the clinical indications for the use of this combination.

Circumferential viscodilation of Schlemm's canal for open-angle glaucoma: ab-interno vs ab-externo canaloplasty with tensioning suture.

PurposeTo compare the efficacy of minimally invasive ab-interno canaloplasty (ABiC) vs ab-externo canaloplasty (CP) in reducing intraocular pressure (IOP) and glaucoma medication dependence.Patients and methodsThis nonrandomized, retrospective, single-center, paired eye study assessed the 12-month outcomes of 12 patients with primary open-angle glaucoma who underwent ABiC in one eye and CP in the other eye, either as stand-alone procedures or combined with cataract extraction. Primary endpoints included mean IOP and number of glaucoma medications at 12 months postoperative. Secondary endpoints included surgical complications and secondary interventions.ResultsFour males and eight females with a mean age of 73.8±12.6 years were included. In the CP group, the mean preoperative IOP was 18.1±3.9 mmHg on 2.4±0.5 medications, which reduced to 13.5±2.2 mmHg (P<0.05) on 0.9±0.9 medications (P<0.001). In the ABiC group, the mean preoperative IOP was 18.5±3.4 mmHg on 2.4±0.5 medications and postoperative IOP was 13.8±2.2 mmHg (P<0.05) on 0.8±0.8 medications (P<0.05). There was no significant difference in IOP and medication use between treatment groups at 12 months postoperative. No serious adverse events were recorded in either group, though two patients in the CP group developed pressure spikes 10 mmHg beyond preoperative IOP.ConclusionThis paired eye study found ABiC to have comparable IOP lowering and glaucoma medication reduction to CP in open-angle glaucoma. This suggests ABiC may be a suitable method for improving aqueous outflow via the trabecular pathway. Further large-scale investigation is needed.

Rho- Kinase Inhibitor Y-27632 Decreases the Thickness of Trabecular Meshwork in Juvenile Rats Mod-el Injected with Sodium Hyaluronate

Most glaucoma drugs lower the intraocular pressure (IOP) by decreasing the aqueous humor production and increasing the outflow through uveoscleral pathway. None of these drugs work mainly on increasing outflow through the trabecular pathway. Consequently, the experiment to develop glaucoma drugs directly target at the trabecular outflow pathway is highly required. The purpose of this study is to reveal the effect of rho-kinase inhibitor Y-27632 on the thickness of trabecular meshwork in juvenile rats model injected with sodium hyalu-ronate. This study was an experimental study with posttest only control group design. Twenty-four rats were included in this study. Each eye of the rat would be considered as one sample. Samples were divided into 6 groups, negative control group, positive control I group with intracameral sodium hyaluronate injection, posi-tive control II group with topical Y-27632 10 mM, and three experimental groups with intracameral injection of sodium hyaluronate and Y-27632 10-1 mM, 1 mM, and 10 mM respectively. After the procedures all rats were sacrificed and enucleated. Trabecular meshwork tissue was stained with Hematoxilene-Eosin and evalu-ated under 400× microscopic magnification. Quantitative measurements were taken using computerized image analysis with dot slide program. There were significant statistic differences among the positive control I group and the experimental groups (p-value < 0.05) as well as the positive control II group and the experi-mental groups (p-value < 0.05). The highest mean of decreasing trabecular meshwork thickness was noted in the group given by sodium hyaluronate and Y-27632 10 mM with value of 118.42 µm. There was decreasing thickness of trabecular meshwork due to the effect of rho- kinase inhibitor Y-27632 in juvenile rats injected with sodium hyaluronate.

Microinvasive glaucoma surgeries

Because of the complications of current penetrating glaucoma surgeries as in trabeculectomy or glaucoma drainage devices, there is now an increased interest in newly introduced microinvasive glaucoma surgeries, which provide a safer and not a less-efficient alternative for such procedures, along with a decrease in dependence of glaucoma medications postoperatively. The microscopic implants can be implanted by following either an ab-interno approach (e.g. iStent, CyPass, Trabectome, and Xen Gel microshunt) or an ab-externo approach (e.g. Ex-Press minishunt and canaloplasty). The procedures aim to utilize the trabecular pathway, suprachoroidal space, or the subconjunctival space to increase aqueous drainage. These procedures can be performed in conjunction with phacoemulsification to augment the intraocular pressure-lowering effect, and the devices can be implanted during the same session without or with minimal additional risk.

Advances in Glaucoma Surgery

Currently popular glaucoma filtration surgeries which include trabeculectomy and glaucoma drainage devices have their share of vision-threatening complications. Lately less invasive and safer devices have been developed to be used alone or in conjunction with cataract surgery for lowering the intraocular pressure. These microscopic implants utilize the trabecular pathway, suprachoroidal pathway or subconjunctival route for drainage. The term ‘Micro-Invasive Glaucoma Surgery’ includes surgical procedures that use an ab-interno approach, such as iStent, Trabectome, Hydrus Scaffold shunt, Excimer laser trabeculotomy and CyPass suprachoroidal shunt. Implants which utilize ab-externo approach are canaloplasty with microcatheter, Ex-PRESS glaucoma microshunts, SOLX Gold shunt and Aquashunt. Studies are ongoing to evaluate their efficacy alone or in combination with phacoemulsification and have found encouraging results in early to moderate glaucoma.

Elevated intraocular pressure induces Rho GTPase mediated contractile signaling in the trabecular meshwork

Rho GTPase regulated contractile signaling in the trabecular meshwork (TM) has been shown to modulate aqueous humor (AH) outflow and intraocular pressure (IOP). To explore whether elevated IOP, a major risk factor for primary open angle glaucoma (POAG) influences Rho GTPase signaling in the TM, we recorded AH outflow in enucleated contralateral porcine eyes perfused for 4–5 h at either 15 mm or 50 mm Hg pressure. After perfusion, TM tissue extracted from perfused eyes was evaluated for the activation status of Rho GTPase, myosin light chain (MLC), myosin phosphatase target substrate 1 (MYPT1), myristoylated alanine-rich C-kinase substrate (MARCKS) and paxillin. Eyes perfused at 50 mm Hg exhibited a significant decrease in AH outflow facility compared with those perfused at 15 mm Hg. Additionally, TM tissue from eyes perfused at 50 mm Hg revealed significantly increased levels of activated RhoA and phosphorylated MLC, MYPT1, MARCKS and paxillin compared to TM tissue derived from eyes perfused at 15 mm Hg. Taken together, these observations indicate that elevated IOP-induced activation of Rho GTPase-dependent contractile signaling in the TM is associated with increased resistance to AH outflow through the trabecular pathway, and demonstrate the sensitivity of Rho GTPase signaling to mechanical force in the AH outflow pathway.

Autotaxin-Lysophosphatidic Acid Axis Is a Novel Molecular Target for Lowering Intraocular Pressure

Primary open-angle glaucoma is the second leading cause of blindness in the United States and is commonly associated with elevated intraocular pressure (IOP) resulting from diminished aqueous humor (AH) drainage through the trabecular pathway. Developing effective therapies for increased IOP in glaucoma patients requires identification and characterization of molecular mechanisms that regulate IOP and AH outflow. This study describes the identification and role of autotaxin (ATX), a secretory protein and a major source for extracellular lysophosphatidic acid (LPA), in regulation of IOP in a rabbit model. Quantitative proteomics analysis identified ATX as an abundant protein in both human AH derived from non-glaucoma subjects and in AH from different animal species. The lysophospholipase D (LysoPLD) activity of ATX was found to be significantly elevated (by ∼1.8 fold; n = 20) in AH derived from human primary open angle glaucoma patients as compared to AH derived from age-matched cataract control patients. Immunoblotting analysis of conditioned media derived from primary cultures of human trabecular meshwork (HTM) cells has confirmed secretion of ATX and the ability of cyclic mechanical stretch of TM cells to increase the levels of secreted ATX. Topical application of a small molecular chemical inhibitor of ATX (S32826), which inhibited AH LysoPLD activity in vitro (by >90%), led to a dose-dependent and significant decrease of IOP in Dutch-Belted rabbits. Single intracameral injection of S32826 (∼2 µM) led to significant reduction of IOP in rabbits, with the ocular hypotensive response lasting for more than 48 hrs. Suppression of ATX expression in HTM cells using small-interfering RNA (siRNA) caused a decrease in actin stress fibers and myosin light chain phosphorylation. Collectively, these observations indicate that the ATX-LPA axis represents a potential therapeutic target for lowering IOP in glaucoma patients.

The progress of aqueous humor outflow pathway in open-angle glaucoma

Primary open angle glaucoma (POAG) is defined as an optic neuropathy characterized by loss of optic nerve axons and retinal ganglion cells. Increase of intraocular pressure (IOP) is a major risk factor for POAG, which is partially due to an abnormally high aqueous humor outflow resistance in trabecular meshwork (TM). Trabecular outflow pathway is a key element in drainage of aqueous humor. Malfunction of TM in POAG is associated with over expression of inflammatory markers, cellular senescence, oxidative stress, and decrease of cellularity. The biological changes of TM cell and extracellular matrix compromise trabecular pathway drainage resulting in an increased IOP. This article reviewed the progression in research on aqueous humor outflow pathway emphasizing the alteration of trabecular pathway.

Mechanistic basis of Rho GTPase-induced extracellular matrix synthesis in trabecular meshwork cells

Elevated intraocular pressure arising from impaired aqueous humor drainage through the trabecular pathway is a major risk factor for glaucoma. To understand the molecular basis for Rho GTPase-mediated resistance to aqueous humor drainage, we investigated the possible interrelationship between actomyosin contractile properties and extracellular matrix (ECM) synthesis in human trabecular meshwork (TM) cells expressing a constitutively active form of RhoA (RhoAV14). TM cells expressing RhoAV14 exhibited significant increases in fibronectin, tenascin C, laminin, alpha-smooth muscle actin (alpha-SMA) levels, and matrix assembly in association with increased actin stress fibers and myosin light-chain phosphorylation. RhoAV14-induced changes in ECM synthesis and actin cytoskeletal reorganization were mimicked by lysophosphatidic acid and TGF-beta(2), known to increase resistance to aqueous humor outflow and activate Rho/Rho kinase signaling. RhoAV14, lysophosphatidic acid, and TGF-beta(2) stimulated significant increases in Erk1/2 phosphorylation, paralleled by profound increases in fibronectin, serum response factor (SRF), and alpha-SMA expression. Treatment of RhoA-activated TM cells with inhibitors of Rho kinase or Erk, on the other hand, decreased fibronectin and alpha-SMA levels. Although suppression of SRF expression (both endogenous and RhoA, TGF-beta(2)-stimulated) via the use of short hairpin RNA decreased alpha-SMA levels, fibronectin was unaffected. Conversely, fibronectin induced alpha-SMA expression in an SRF-dependent manner. Collectively, data on RhoA-induced changes in actomyosin contractile activity, ECM synthesis/assembly, and Erk activation, along with fibronectin-induced alpha-SMA expression in TM cells, reveal a potential molecular interplay between actomyosin cytoskeletal tension and ECM synthesis/assembly. This interaction could be significant for the homeostasis of aqueous humor drainage through the pressure-sensitive trabecular pathway.

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells.

Percolation theory relates corticocancellous architecture to mechanical function in vertebrae of inbred mouse strains

Complex corticocancellous skeletal sites such as the vertebra or proximal femur are connected networks of bone capable of transferring mechanical loads. Characterizing these structures as networks may allow us to quantify the load transferring behavior of the emergent system as a function of the connected cortical and trabecular components. By defining the relationship between certain physical bone traits and mechanical load transfer pathways, a clearer picture of the genetic determinants of skeletal fragility can be developed. We tested the hypothesis that the measures provided by network percolation theory will reveal that different combinations of cortical, trabecular, and compositional traits lead to significantly different load transfer pathways within the vertebral bodies among inbred mouse strains. Gross morphologic, micro-architectural, and compositional traits of L5 vertebrae from 15 week old A/J (A), C57BL6/J (B6), and C3H/HeJ (C3H) inbred mice ( n = 10/strain) were determined using micro-computed tomography. Measures included total cross-sectional area, bone volume fraction, trabecular number, thickness, spacing, cortical area, and tissue mineral density. Two-dimensional coronal sections were converted to network graphs with the cortical shell considered as one highly connected node. Percolation parameters including correlation length (average number of connected nodes between superior and inferior surfaces), chemical length (minimum number of connected nodes between surfaces), and backbone mass (strut number) were measured. Analysis of the topology of the connected bone networks showed that A and B6 mice transfer load through trabecular pathways in the middle of the vertebral body in addition to the cortical shell. C3H mice transfer load primarily through the highly mineralized cortical shell. Thus, the measures provided by percolation theory provide a quantitative approach to study how different combinations of cortical and trabecular traits lead to mechanically functional structures. The data further emphasize the interdependent nature of these physical bone traits suggesting similar genetic variants may affect both trabecular and cortical bone. Therefore, developing a network approach to study corticocancellous architecture during growth should further our understanding of the biological basis of skeletal fragility and, thus, provide novel engineering approaches to studying the genetic basis of fracture risk.

Studies on the mechanism of action of timolol and on the effects of suppression and redirection of aqueous flow on outflow facility

Long-term use of drugs that suppress aqueous humor formation, such as timolol and dorzolamide, or that redirect aqueous humor outflow from the trabecular meshwork, such as prostaglandin F 2α analogues, could cause underperfusion of the trabecular meshwork and a secondary decrease in outflow facility. We investigated the mechanism of suppression of aqueous humor formation by timolol in monkey eyes by measuring aqueous humor ascorbate levels. We also determined whether suppression of aqueous humor formation with and without redirection of aqueous humor away from the trabecular meshwork could lead to a subsequent reduction in outflow facility, and whether this reduction was correlated with increased fibronectin levels in anterior chamber aqueous humor. In cynomolgus monkeys, unilateral dose/aqueous humor formation response curves were generated for timolol, dorzolamide, and a combination of timolol+dorzolamide. Aqueous humor formation and/or outflow facility were measured in both eyes after approximately four days, four weeks and seven weeks of twice daily treatment with 3·5 μg timolol+1·0 mg dorzolamide to one eye and 30% DMSO to the other. In some monkeys, 5 μg prostaglandin F 2α-isopropyl ester (PG) was added to timolol+dorzolamide for 4-week treatments. Intraocular pressure and corneal endothelial transfer coefficients ( k a) were also measured at four weeks. Aqueous humor fibronectin levels were determined in four monkeys after approximately 9·5 weeks of timolol+dorzolamide treatment. Aqueous humor formation, intraocular pressure, and aqueous humor ascorbate levels were also determined in rhesus monkeys at baseline and after a single unilateral topical administration of 25 μg timolol. Compared to baseline for the same eye, aqueous humor formation was significantly decreased in treated eyes at all doses of timolol and at 1·8 and 4 mg dorzolamide. Compared to the opposite control eye, aqueous humor formation was lower in treated eyes after 3·5 and 5 μg timolol and after all doses of dorzolamide. Aqueous humor formation after treatment with 3·5 μg timolol+1·0 mg dorzolamide was decreased in treated vs. control eyes, after four days and was suppressed in both treated and control eyes after four weeks of treatment, but not when PG was added. There was no difference in k a values with or without the addition of PG. Intraocular pressure was significantly lower in both treated and control eyes vs. baseline after ∼6·5 weeks treatment with timolol+dorzolamide when taken 2 hr after the last dose and after ∼3·5 weeks treatment with timolol+dorzolamide+PG when measured 6 hr after the last dose. Outflow facility after treatment with timolol+dorzolamide was unchanged after four days, tended to be lower in the treated vs. control eyes after four and seven weeks, and was significantly lower in treated vs. control eyes after four weeks treatment with timolol+dorzolamide+PG (0·352±0·052 vs. 0·515±0·096 μl min −1 mmHg −1, p≤0·02). Both treated vs. control eye aqueous humor fibronectin levels were below the level of detection for our assay (0·01 μg ml −1). The 25 μg timolol dose decreased ipsilateral, but not contralateral intraocular pressure (12·6±1·7 vs. 15·2±0·9; p<0·05) and aqueous humor formation (1·40±0·08 vs. 2·03±0·09 μg ml −1, p≤0·01). There was no difference in anterior chamber ascorbate levels in treated vs. control eyes or compared to their respective baselines. Our findings indicate that timolol affects neither ciliary epithelial transport of ascorbate nor aqueous fibronectin levels. Our data also indicate that decreasing aqueous humor formation over a period of time can lead to reduction in outflow facility, particularly when combined with therapy that redirects aqueous from the trabecular meshwork. Future intraocular pressure-lowering therapies for glaucoma may better be directed at enhancing flow through the trabecular pathway as opposed to decreasing aqueous humor formation or rerouting aqueous humor away from the trabecular meshwork.

Recent observations concerning the effects of cholinergic drugs on outflow facility in monkeys

Several major areas of work by the author and his international collaborators are reviewed. (1) The ciliary muscle in the non-human primate eye was disinserted at the scleral spur. Pilocarpine was then ineffective in increasing outflow facility, indicating that ciliary muscle contraction mediated the IOP-lowering effect of muscarinic cholinergics. (2) Compounds such as cytochalasins, H-7 and latrunculin A/B, which alter the actin cytoskeleton, cellular contractility and cellular adhesions in cultured trabecular meshwork cells, relaxed trabecular pathway cells and consequently the meshwork itself so as to decrease IOP and enhance trabecular outflow facility in non-human primates. Gene transfer approaches utilizing C3 and caldesmon over-expression by viral vectors to target specific steps in the cellular contractility/cytoskeleton/cell adhesion cascades characteristically altered trabecular meshwork cell morphology and increased outflow facility in organ-cultured anterior segments. (3) Prostaglandin F2α analogues enhanced matrix metalloproteinase production by ciliary muscle cells and scleral fibroblasts, leading to remodeling of the extracellular matrix of the ciliary muscle and sclera and consequently to increased uveoscleral outflow and decreased IOP in primates. (4) The rhesus monkey was an excellent model for human presbyopia, losing the accommodative response to cholinergic stimulation in the same timeframe relative to lifespan. No changes were found in ciliary muscle enzymes involved in acetylcholine biosynthesis or degradation or in muscarinic receptor numbers or affinity. Contractility of isolated ciliary muscle did not diminish with age, but posterior ciliary muscle attachments stiffened, suggesting a possible role in restricting muscle and consequently lens movement during accommodation. A model to reproducibly stimulate accommodation through central stimulation of the Edinger–Westphal nucleus was developed. Goniovideography and ultrasound biomicroscopic techniques allowed real-time recording and analysis of the accommodation-relevant structures. Surgical ablation of the intraocular structures involved in the accommodation response has led to further understanding of their roles and changes with age related to presbyopia. (5) Global collaborations such as those involved in these studies will be essential in the future, as science becomes “bigger”.