Consensus Recommendations for Studies of Outflow Facility and Intraocular Pressure Regulation Using Ex Vivo Perfusion Approaches.

Intraocular pressure (IOP) elevation is the primary risk factor and currently the main treatable factor for progression of glaucomatous optic neuropathy. In addition to direct clinical and living animal in vivo studies, ex vivo perfusion of anterior segments and whole eyes is a key technique for studying conventional outflow function as it is responsible for IOP regulation. We present well-tested experimental details, protocols, considerations, advantages, and limitations of several ex vivo model systems for studying IOP regulation.

Consensus Statement on Brillouin Light Scattering Microscopy of Biological Materials.

Brillouin Light Scattering (BLS) spectroscopy is a non-invasive, non-contact, label-free optical technique that can provide information on the mechanical properties of a material on the sub-micron scale. Over the last decade it has seen increased applications in the life sciences, driven by the observed significance of mechanical properties in biological processes, the realization of more sensitive BLS spectrometers and its extension to an imaging modality. As with other spectroscopic techniques, BLS measurements not only detect signals characteristic of the investigated sample, but also of the experimental apparatus, and can be significantly affected by measurement conditions.

Endothelial cell stiffness and type drive the formation of biomechanically-induced transcellular pores.

Formation of transcellular pores facilitates the transport of materials across endothelial barriers. In Schlemm's canal (SC) endothelium, impaired pore formation is associated with glaucoma. However, our understanding of the cellular processes responsible for pore formation is limited by lack of assays.

The Factors Affecting the Stability of IOP Homeostasis.

Purpose: Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions).

Pressure Clamping During Ocular Perfusions Drives Nitric Oxide-Mediated Washout.

Purpose: The aim of this study was to test the hypothesis that nitric oxide (NO) mediates a pressure-dependent, negative feedback loop that maintains conventional outflow homeostasis and thus IOP. If true, holding pressure during ocular perfusions will result in uncontrolled production of NO, hyper-relaxation of the trabecular meshwork, and washout.

Methods: Paired porcine eyes were perfused at constant pressure of 15 mm Hg.

Model of inverse bleb growth explains giant vacuole dynamics during cell mechanoadaptation.

Cells can withstand hostile environmental conditions manifest as large mechanical forces such as pressure gradients and/or shear stresses by dynamically changing their shape. Such conditions are realized in the Schlemm's canal of the eye where endothelial cells that cover the inner vessel wall are subjected to the hydrodynamic pressure gradients exerted by the aqueous humor outflow. These cells form fluid-filled dynamic outpouchings of their basal membrane called .

Segmental outflow dynamics in the trabecular meshwork of living mice.

Aqueous humour does not drain uniformly through the trabecular meshwork (TM), but rather follows non-uniform or "segmental" routes. In this study, we examined whether segmental outflow patterns in the TM change over time in living mice and whether such changes are affected by age. Segmental outflow patterns were labelled by constant-pressure infusion of fluorescent tracer microparticles into the anterior chamber of anesthetised C57BL/6J mice at 3 or 8 months of age.

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.

Consensus Recommendation for Mouse Models of Ocular Hypertension to Study Aqueous Humor Outflow and Its Mechanisms.

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.

and Are Putative Modulators of Corneoscleral Stiffness.

The biomechanical properties of the cornea and sclera are important in the onset and progression of multiple ocular pathologies and vary substantially between individuals, yet the source of this variation remains unknown. Here we identify genes putatively regulating corneoscleral biomechanical tissue properties by conducting high-fidelity ocular compliance measurements across the BXD recombinant inbred mouse set and performing quantitative trait analysis. We find seven cis-eQTLs and non-synonymous SNPs associating with ocular compliance, and show by RT-qPCR and immunolabeling that only two of the candidate genes, and , showed significant expression in corneal and scleral tissues.

The role of Piezo1 in conventional aqueous humor outflow dynamics.

Controlling intraocular pressure (IOP) remains the mainstay of glaucoma therapy. The trabecular meshwork (TM), the key tissue responsible for aqueous humor (AH) outflow and IOP maintenance, is very sensitive to mechanical forces. However, it is not understood whether Piezo channels, very sensitive mechanosensors, functionally influence AH outflow.

The ocular pulse decreases aqueous humor outflow resistance by stimulating nitric oxide production.

Intraocular pressure (IOP) is not static, but rather oscillates by 2-3 mmHg because of cardiac pulsations in ocular blood volume known as the ocular pulse. The ocular pulse induces pulsatile shear stress in Schlemm's canal (SC). We hypothesize that the ocular pulse modulates outflow facility by stimulating shear-induced nitric oxide (NO) production by SC cells.

Deletion of the gene encoding prostamide/prostaglandin F synthase reveals an important role in regulating intraocular pressure.

Prostamide/prostaglandin F synthase (PM/PGFS) is an enzyme with very narrow substrate specificity and is dedicated to the biosynthesis of prostamide F and prostaglandin F (PGF). The importance of this enzyme, relative to the aldo-keto reductase (AKR) series, in providing functional tissue prostamide F levels was determined by creating a line of PM/PGFS gene deleted mice. Deletion of the gene encoding PM/PGFS (Fam213b / Prxl2b) was accomplished by a two exon disruption.

siRNA targeting Schlemm's canal endothelial tight junctions enhances outflow facility and reduces IOP in a steroid-induced OHT rodent model.

Systemic or localized application of glucocorticoids (GCs) can lead to iatrogenic ocular hypertension, which is a leading cause of secondary open-angle glaucoma and visual impairment. Previous work has shown that dexamethasone increases zonula occludens-1 (ZO-1) protein expression in trabecular meshwork (TM) cells, and that an antisense oligonucleotide inhibitor of ZO-1 can abolish the dexamethasone-induced increase in -endothelial flow resistance in cultured Schlemm's canal (SC) endothelial and TM cells. We have previously shown that intracameral inoculation of small interfering RNA (siRNA) targeting SC endothelial cell tight junction components, ZO-1 and tricellulin, increases aqueous humor outflow facility in normotensive mice by reversibly opening SC endothelial paracellular pores.

The vital role for nitric oxide in intraocular pressure homeostasis.

Catalyzed by endothelial nitric oxide (NO) synthase (eNOS) activity, NO is a gaseous signaling molecule maintaining endothelial and cardiovascular homeostasis. Principally, NO regulates the contractility of vascular smooth muscle cells and permeability of endothelial cells in response to either biochemical or biomechanical cues. In the conventional outflow pathway of the eye, the smooth muscle-like trabecular meshwork (TM) cells and Schlemm's canal (SC) endothelium control aqueous humor outflow resistance, and therefore intraocular pressure (IOP).

The role of EP receptors in mediating the ultra-long-lasting intraocular pressure reduction by JV-GL1.

Background: A single application of JV-GL1 substantially lowers non-human primate intraocular pressure (IOP) for about a week, independent of dose. This highly protracted effect does not correlate with its ocular biodisposition or correlate with the once-daily dosing regimen for other prostanoid EP receptor agonists such as trapenepag or omidenepag. The underlying pharmacological mechanism for the multiday extended activity of JV-GL1 is highly intriguing.

Infusion Mechanisms in Brain White Matter and Their Dependence on Microstructure: An Experimental Study of Hydraulic Permeability.

Hydraulic permeability is a topic of deep interest in biological materials because of its important role in a range of drug delivery-based therapies. The strong dependence of permeability on the geometry and topology of pore structure and the lack of detailed knowledge of these parameters in the case of brain tissue makes the study more challenging. Although theoretical models have been developed for hydraulic permeability, there is limited consensus on the validity of existing experimental evidence to complement these models.

Aqueous Humor Outflow Requires Active Cellular Metabolism in Mice.

Purpose: Conventional wisdom posits that aqueous humor leaves the eye by passive bulk flow without involving energy-dependent processes. However, recent studies have shown that active processes, such as cell contractility, contribute to outflow regulation. Here, we examine whether inhibiting cellular metabolism affects outflow facility in mice.

Age and Menopause Effects on Ocular Compliance and Aqueous Outflow.

Purpose: Glaucoma is the second leading cause of blindness worldwide. Recent work suggests that estrogen and the timing of menopause play a role in modulating the risk of developing glaucoma. Menopause is known to cause modest changes in intraocular pressure; yet, whether this change is mediated through the outflow pathway remains unknown.

Shear Stress in Schlemm's Canal as a Sensor of Intraocular Pressure.

Elevated intraocular pressure (IOP) narrows Schlemm's canal (SC), theoretically increasing luminal shear stress. Using engineered adenoviruses containing a functional fragment of the shear-responsive endothelial nitric oxide synthase (eNOS) promoter, we tested effects of shear stress and elevated flow rate on reporter expression in vitro and ex vivo. Cultured human umbilical vein endothelial cells (HUVECs) and SC cells were transduced with adenovirus containing eNOS promoter driving secreted alkaline phosphatase (SEAP) or green fluorescent protein (GFP) and subjected to shear stress.

The β4-Subunit of the Large-Conductance Potassium Ion Channel KCa1.1 Regulates Outflow Facility in Mice.

Purpose: The large-conductance calcium-activated potassium channel KCa1.1 (BKCa, maxi-K) influences aqueous humor outflow facility, but the contribution of auxiliary β-subunits to KCa1.1 activity in the outflow pathway is unknown.

Measurement of Ocular Compliance Using iPerfusion.

The pressure-volume relationship of the eye is determined by the biomechanical properties of the corneoscleral shell and is classically characterised by Friedenwald's coefficient of ocular rigidity or, alternatively, by the ocular compliance (OC), defined as dV/dP. OC is important in any situation where the volume (V) or pressure (P) of the eye is perturbed, as occurs during several physiological and pathological processes. However, accurately measuring OC is challenging, particularly in rodents.

Ultrastructural variability of the juxtacanalicular tissue along the inner wall of Schlemm's canal.

Purpose: Increased resistance of aqueous humor drainage from the eye through Schlemm's canal (SC) is the basis for elevated intraocular pressure in glaucoma. Experimental evidence suggests that the bulk of outflow resistance lies in the vicinity of the inner wall endothelial lining of SC and the adjacent juxtacanalicular tissue (JCT). However, there is little understanding of how this resistance is generated, and a detailed understanding of the structure-function relationship of the outflow pathway has not been established yet.

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

Mice are routinely used to study aqueous humour dynamics. However, physical factors such as temperature and hydration affect outflow facility in enucleated eyes. This retrospective study examined whether differences in temperature and relative humidity experienced by living mice within their housing environment in vivo coincide with differences in outflow facility measured ex vivo.

Detection of proteoglycan loss from articular cartilage using Brillouin microscopy, with applications to osteoarthritis.

The degeneration of articular cartilage (AC) occurs in osteoarthritis (OA), which is a leading cause of pain and disability in middle-aged and older people. The early disease-related changes in cartilage extra-cellular matrix (ECM) start with depletion of proteoglycan (PG), leading to an increase in tissue hydration and permeability. These early compositional changes are small (<10%) and hence difficult to register with conventional non-invasive imaging technologies (magnetic resonance and ultrasound imaging).