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

Purpose: NCX 667, a novel nitric oxide (NO) donor with an isomannide core, was characterized for its IOP-lowering ability in animal models of ocular hypertension and glaucoma. Bioengineered human trabecular meshwork/Schlemm's canal (HTM/HSC) constructs were used to explore the mode of action., Methods: Ocular normotensive New Zealand white (NZW) rabbits (ONT-rabbits), spontaneously ocular hypertensive pigmented Dutch-belted rabbits (sOHT-rabbits), hypertonic saline (5%)-induced transient ocular hypertensive NZW rabbits (tOHT-rabbits), ocular normotensive Beagle dogs (ONT-dogs), and laser-induced ocular hypertensive cynomolgus monkeys (OHT-monkeys) were used. NCX 667 or vehicle (30 µL) was instilled in a crossover, masked fashion and intraocular pressure (IOP) measured before dosing (baseline) and for several hours thereafter. The ONT-rabbits were used for cyclic guanosine monophosphate (cGMP) determination in ocular tissues after ocular dosing with NCX 667. Transforming growth factor-beta2 (TGFβ2) (2.5 ng/mL, six days)-treated HTM/HSC constructs were used to address changes in outflow facility., Results: NCX 667 resulted in robust and dose-dependent IOP decrease in all models used. Maximal IOP-lowering efficacy at 1% was -4.1 ± 0.6, -12.2 ± 2.7, -10.5 ± 2.0, -5.3 ± 0.8, and -6.6 ± 1.9 mmHg, respectively, in ONT-dogs, sOHT-rabbits, tOHT-rabbits, ONT-rabbits, and OHT-monkeys. In ONT-rabbits NCX 667 (1%) increased cGMP in aqueous humor (AH) but not in retina and iris/ciliary body. NCX 667 concentration-dependently increased outflow facility in TGFβ2-treated HTM/HSC constructs (outflow facility, 0.10 ± 0.06 and 0.30 ± 0.10 µL/min/mmHg/mm2, respectively, in vehicle- and NCX 667-treated constructs)., Conclusions: NCX 667 leads to robust IOP lowering in several animal models. Evidence in HTM/HSC constructs indicate that the IOP reduction likely results from NO-mediated increase of the conventional outflow pathway. Other mechanisms including changes in AH production and episcleral vein pressure may not be excluded at this time.

Purpose: To study the effect of 3 Schlemm's canal (SC) microinvasive glaucoma surgery (MIGS) devices on outflow facility., Design: Paired comparisons, randomized design, baseline-controlled study., Participants: Thirty-six pairs of dissected anterior segments from donated human eye bank eyes without glaucoma were studied. A baseline measurement was collected from each eye to serve as its control., Methods: Using a constant pressure perfusion method, outflow facility was measured in paired eyes from human donors. Measurements were made at perfusion pressures of 10 mmHg, 20 mmHg, 30 mmHg, and 40 mmHg. Outflow facility was measured before (baseline control) and after the implantation of an SC glaucoma drainage device or sham procedure. Three sets of experiments were carried out comparing 1 and 2 iStent Trabecular Micro-Bypass Stents and 2 iStent Inject implants with the Hydrus Microstent., Main Outcome Measures: Change in outflow facility from baseline or contralateral eye., Results: After Hydrus placement, the outflow facility increased from 0.23±0.03 μl/minute per millimeter of mercury at baseline to 0.38±0.03 μl/minute per millimeter of mercury (P < 0.001). The percent increase in outflow facility was 79±21% for the Hydrus and 11±16% for the 2 iStent Inject devices, a difference that was significant (P = 0.018). Outflow facility with 1 iStent (0.38±0.07 μl/minute per millimeter of mercury) was greater than baseline (0.28±0.03 μl/minute per millimeter of mercury; P = 0.031). The 1 iStent showed a greater increase in outflow facility from baseline (0.10±0.04 μl/minute per millimeter of mercury) compared with the sham procedure (-0.08±0.05 μl/minute per millimeter of mercury; P = 0.042). No other significant differences were found., Conclusions: The longer the MIGS device, and thus the more SC that it dilates, the greater the outflow facility., (Copyright © 2019 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Purpose: Hyposecretion of aqueous humor has been postulated to adversely affect the health of the trabecular meshwork and outflow resistance. However, the effect of medications that reduce aqueous humor production on outflow facility in living human eyes is unclear. This study evaluated the effect of timolol, an aqueous humor flow suppressant, on outflow facility in healthy eyes., Design: Prospective, before-and-after study., Methods: In a multicenter study, 113 healthy participants over 40 years of age were included. Intraocular pressure (IOP) was measured with the participant in the sitting position by using a pneumatonometer. The outflow facility was measured with the participant in the supine position by 2-minute pneumatonography. After participants self-administered drops of timolol 0.5% for 1 week, twice daily in each eye, both measurements were repeated., Results: Mean IOP decreased from 15.1 ± 3.0 mm Hg at baseline to 12.4 ± 2.4 mm Hg (P < 0.001) after 1 week of timolol use. Mean outflow facility decreased from 0.23 ± 0.08 μL/min/mm Hg at baseline to 0.18 ± 0.08 μL/min/mm Hg (P < 0.001) after timolol. The change in outflow facility was negatively correlated with baseline outflow facility (r = -0.51; P < 0.001)., Conclusions: Timolol reduces outflow facility in healthy human eyes, and this effect is greater in eyes with higher baseline outflow facility. This phenomenon may be related to reduced aqueous humor flow, but the precise mechanism remains to be determined., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: To determine improvement in outflow facility (C) in human anterior segments implanted with a novel Schlemm's canal scaffold or two trabecular micro-bypasses., Methods: Human anterior segments were isolated from 12 pairs of eyes from donors with no history of ocular disease and then perfused at 50, 40, 30, 20, and 10 mm Hg pressures for 10 minutes each. Baseline C was calculated from perfusion pressures and flow rates. The scaffold was implanted into Schlemm's canal of one anterior segment, and two micro-bypasses were implanted three clock-hours apart in the contralateral anterior segment. Outflow facility and resistance were compared at various standardized perfusion pressures and between each device., Results: Compared to baseline, C increased by 0.16 ± 0.12 μL/min/mm Hg (74%) with the scaffold, and 0.08 ± 0.12 μL/min/mm Hg (34%) with two micro-bypasses. The scaffold increased C at perfusion pressures of 50, 40, 30, and 20 mm Hg (P < 0.005). Two micro-bypasses increased C at a perfusion pressure of 40 mm Hg (P < 0.05)., Conclusions: Both implants effectively increased C in human eyes ex vivo. The scaffold increased C by a greater percentage (73% vs. 34%) and at a greater range of perfusion pressures (20 to 50 mm Hg vs. 40 mm Hg) than the two micro-bypasses, suggesting that the 8-mm dilation of Schlemm's canal by the scaffold may have additional benefits in lowering the outflow resistance. The Hydrus Microstent scaffold may be an effective therapy for increasing outflow facility and thus reducing the IOP in patients with glaucoma.

This study evaluates the morphologic effect of the implantation of two different sizes of the Hydrus microstent on the outer wall of Schlemm's canal (SC) and collector channel (CC) ostia. Twelve human eyes were dissected at the equator removing the iris, lens, ciliary body and vitreous. The cornea was excised with a corneal trephine exposing a direct view of the angle while leaving the trabecular meshwork (TM) intact. The Hydrus delivery system was used to deliver microstents of 8 mm and 15 mm in length into SC. Following delivery, the tissues were immediately immersed in fixative. After tissue fixation, the microstents were gently lifted out of SC through the TM leaving a small slit opening in the TM. The slit opening was widened by gently dissecting the entire TM. Control eyes underwent dissection before fixation by gently removing the TM exposing the outer wall of SC. The tissues were prepared for scanning electron microscopy (SEM). The external wall of SC was imaged using SEM and were reviewed with particular attention focused on the distribution of irregular particulate matter (IPM), the shape of the CC ostia and the health of the SC endothelium. Three eyes received the 8 mm microstent, two the 15 mm microstent and 6 eyes served as controls. Five of the controls had reported histories of glaucoma while all other eyes were normal. All eyes showed evidence of removal of the trabecular meshwork revealing the external wall of SC. CCs were regularly visible in all eyes and were not obstructed, compressed or their margins disrupted. Nuclear profiles were oriented circumferentially in SC except at regions of CC ostia where they assumed a radial configuration oriented toward the lumen of the CC. The area of microstent contact with SC external wall was examined with SEM and a comparison made between the 8 and 15 mm microstent showing a smaller area of indentation with the 8 mm microstent. The indentations were generally free of particulate debris, were smooth and were devoid of nuclear profiles. In bridged areas adjacent to areas of microstent contact, CCs were identified, appearing patent and intact like those of the control eyes. The eyes receiving 8 mm and 15 mm Hydrus microstents both maintained CC ostia patency but a smaller area of external wall contact was evident from insertion of the 8 mm microstent., (Copyright © 2014. Published by Elsevier Ltd.)

Purpose: The study of aqueous humor dynamics (AHD) in mice is becoming more prevalent as more strains with elevated intraocular pressure (IOP) are developed. High IOP is usually associated with reduced outflow facility making this one of the more important AHD parameters to evaluate. Ocular measurements in mice require anesthesia that has profound effects on IOP but unknown effects on outflow facility. This study evaluates the effects of anesthesia duration and latanoprost treatment on outflow facility and IOP in BALB/c mice., Methods: IOPs were measured in conscious and anesthetized mice by tonometry. Outflow facility was evaluated in 15-min intervals at three pressure levels over two 45-min periods. Comparisons were made between latanoprost-treated eyes and untreated contralateral eyes. To determine the effect of anesthesia duration on IOP, a microneedle method was used to follow IOP for 120 min in separate mice., Results: IOP was 9.7 +/- 0.3 mmHg (mean +/- SEM) in conscious mice and 7.1 +/- 0.02 within 10 min of anesthesia initiation (p < 0.01). IOP changed significantly between but not within assessment periods. IOP at 75 min was significantly (p = 0.004) reduced compared to IOP at 15 min after initial anesthesia. In control eyes, outflow facility did not change between the two 45-min assessment periods during the 120 min test (p = 0.80). In latanoprost-treated eyes, outflow facility increased compared with control eyes during both assessment periods (p = 0.03). A test of filters in series with known resistance found that the method was sensitive enough to detect a change in outflow facility of 0.001 microl/min/mmHg., Conclusions: Administration of ketamine/xylazine anesthesia for 120 min did not alter outflow facility or lessen the effect of latanoprost on outflow facility in mice as determined by a new analysis system. Accurate IOP measurements must be made within minutes of anesthesia administration but outflow facility measurements can be made with less haste.

This study investigated the relationship between decreased outflow facility (C) and changes in hydrodynamic aqueous humor outflow patterns and morphology in cynomolgus monkey eyes with unilateral chronically elevated intraocular pressure (IOP). Argon laser photocoagulation burns to the trabecular meshwork (TM) were made in one eye of each monkey (N = 3), leaving the contralateral eye as a normotensive control. IOPs were followed by pneumatonometry for 16-70 months. C was measured by fluorophotometry before sacrifice. To label the hydrodynamic patterns of outflow, the eyes were enucleated and perfused with fluorescent microspheres (0.5 microm; 0.002%) at the last pressure measured before death minus 7 mmHg. The eyes were perfusion-fixed at the same pressure. Confocal images were taken along the inner wall (IW) of the Schlemm's canal (SC). The total length (TL) and the filtration length (FL) of the IW decorated by tracers were measured in frontal sections. The average percent effective filtration length (PEFL = FL/TL) was calculated for each eye. Sections exhibiting SC were processed and examined under light and electron microscopy. The average IOP was significantly higher in laser-treated eyes (mean +/- SD = 61.33 +/- 4.16 mmHg) than controls (22.67 +/- 4.16 mmHg, P = 0.002). The average C was 13-fold lower in laser-treated eyes (0.03 +/- 0.02 microl/min/mmHg) than controls (0.39 +/- 0.17 microl/min/mmHg, P = 0.057). By confocal microscopy, in control eyes, SC was open and a segmental distribution of microspheres was found in the TM with a greater concentration near the collector channel (CC) ostia. Much less tracer labeling was seen along SC in laser-treated eyes than control eyes. The average PEFL in controls (47.47 +/- 10.79%) was 6-fold larger than in laser-treated eyes (8.40 +/- 4.81%, P = 0.048). The average distance between the inner and outer wall of SC was 5-fold greater in control eyes (18.99 +/- 6.03 microm) than in laser-treated eyes (3.47 +/- 0.33 microm, P = 0.048). By light microscopy, there was extensive pigmentation throughout the TM, denser extracellular matrix in the JCT region, and most of SC collapsed with focal herniations of the IW and JCT protruding into the CC ostia in laser-treated eyes. By electron microscopy, few or no microspheres were observed in laser-treated areas and the areas with SC collapse. More microspheres were observed near the CC ostia area in non-lasered areas. In conclusion, in the laser-induced glaucoma model, laser damage results in a reduction in the available area for outflow across the IW of SC which contributes to the decrease in C and thus elevation of the IOP. Constriction of SC, caused by the chronic elevation of IOP, further decreases the available area for outflow across the IW which decreases C even more in a vicious cycle. This study suggests that the available area for aqueous humor outflow across the IW of SC may play a role in regulating outflow resistance and maintaining IOP.

Oxidative stress and TGFbeta-induced disturbance of cells and tissues are implicated in initiation and progression of pathophysiology of cells/tissues. Using primary human trabecular meshwork (TM) cells from normal and glaucomatous subjects, this study demonstrated that peroxiredoxin (PRDX) 6, an antioxidant, offsets the deleterious effects of oxidative stress on TM cells by optimizing ROS and TGFbeta levels. An analysis of glaucomatous TM cells revealed a reduced expression of PRDX6 mRNA and protein. Biochemical assays disclosed enhanced levels of ROS, as well as high levels of TGFbetas and these cells expressed elevated extracellular matrix (ECM) and Tsp1 proteins with reduced MMP2; conditions implicated in the pathophysiology of glaucoma. Non-glaucomatous TM cells exposed to TGFbetas/ROS showed similar features as in glaucomatous cells. The abnormalities induced were reversed by delivery of PRDX6. The data provide evidence that oxidative stress-induced abnormality in TM may be related to reduced PRDX6 expression and provide a foundation for antioxidant-based therapeutics for treating glaucoma.

Topical treatments with certain prostaglandins (PGs), including FP receptor agonists, lower intraocular pressure by increasing uveoscleral outflow. Although the precise mechanism for the increased uveoscleral outflow is not known, there appears to be activation of a molecular transduction cascade and an increase in the biosynthesis of certain metalloproteinases. This leads to reduction of extracellular matrix components within the ciliary muscle, iris root, and sclera. It is possible that this reduction of extracellular matrix present within portions of the uveoscleral pathway may contribute to the mechanism of increased uveoscleral outflow. Additional mechanisms that may contribute to the PG-mediated increase of uveoscleral outflow include relaxation of the ciliary muscle, cell shape changes, cytoskeletal alteration, or compaction of the extracellular matrix within the tissues of the uveoscleral outflow pathway. Future studies should clarify the importance of these various responses that may contribute to increased uveoscleral outflow. At present, there is no compelling evidence for a substantial facility-increasing effect on the trabecular meshwork outflow for any of these compounds.