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.

Segmental Aqueous Humor Outflow.

Of the known risk factors for glaucoma, elevated intraocular pressure (IOP), is the primary one. The conventional aqueous humor outflow pathway contains the key source of IOP regulation, which is predominantly the trabecular meshwork (TM). Studies of outflow have demonstrated that the outflow pathway is not uniform around the circumference of the eye but highly segmental with regions of relative high flow (HF) and intermediate or medium flow (IF) and regions of low or no flow (LF).

Comparative analysis of traction forces in normal and glaucomatous trabecular meshwork cells within a 3D, active fluid-structure interaction culture environment.

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.

Real-time line-field optical coherence tomography for cellular resolution imaging of biological tissue.

A real-time line-field optical coherence tomography (LF-OCT) system is demonstrated with image acquisition rates of up to 5000 B-frames or 2.5 million A-lines per second for 500 A-lines per B-frame. The system uses a high-speed low-cost camera to achieve continuous data transfer rates required for real-time imaging, allowing the evaluation of future applications in clinical or intraoperative environments.

Dynamic traction force in trabecular meshwork cells: A 2D culture model for normal and glaucomatous states.

Glaucoma, which is associated with intraocular pressure (IOP) elevation, results in trabecular meshwork (TM) cellular dysfunction, leading to increased rigidity of the extracellular matrix (ECM), larger adhesion forces between the TM cells and ECM, and higher resistance to aqueous humor drainage. TM cells sense the mechanical forces due to IOP dynamic and apply multidimensional forces on the ECM. Recognizing the importance of cellular forces in modulating various cellular activities and development, this study is aimed to develop a 2D in vitro cell culture model to calculate the 3D, depth-dependent, dynamic traction forces, tensile/compressive/shear strain of the normal and glaucomatous human TM cells within a deformable polyacrylamide (PAM) gel substrate.

Segmental biomechanics of the normal and glaucomatous human aqueous outflow pathway.

The conventional aqueous outflow pathway, encompassing the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and inner wall endothelium of Schlemm's canal (SC), governs intraocular pressure (IOP) regulation. This study targets the biomechanics of low-flow (LF) and high-flow (HF) regions within the aqueous humor outflow pathway in normal and glaucomatous human donor eyes, using a combined experimental and computational approach. LF and HF TM/JCT/SC complex tissues from normal and glaucomatous eyes underwent uniaxial tensile testing.

Developing an experimental-computational workflow to study the biomechanics of the human conventional aqueous outflow pathway.

The aqueous humor actively interacts with the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and Schlemm's canal (SC) through a dynamic fluid-structure interaction (FSI) coupling. Despite the fact that intraocular pressure (IOP) undergoes significant fluctuations, our understanding of the hyperviscoelastic biomechanical properties of the aqueous outflow tissues is limited. In this study, a quadrant of the anterior segment from a normal human donor eye was dynamically pressurized in the SC lumen, and imaged using a customized optical coherence tomography (OCT).

Differential effects of caveolin-1 and -2 knockdown on aqueous outflow and altered extracellular matrix turnover in caveolin-silenced trabecular meshwork cells.

Purpose: A single nucleotide polymorphism (SNP) identified between caveolin-1 (CAV1) and caveolin-2 (CAV2) on chromosome 7 is associated with glaucoma. One function of CAVs is endocytosis and recycling of extracellular matrix (ECM) components. Here, we generated CAV-silencing lentivirus to evaluate the effects on ECM turnover by trabecular meshwork (TM) cells and to measure the effect on outflow facility in anterior segment perfusion culture.

Intraocular pressure homeostasis: maintaining balance in a high-pressure environment.

Although glaucoma is a relatively common blinding disease, most people do not develop glaucoma. A robust intraocular pressure (IOP) homeostatic mechanism keeps ocular pressures within relatively narrow acceptable bounds throughout most peoples' lives. The trabecular meshwork and/or Schlemm's canal inner wall cells respond to sustained IOP elevation and adjust the aqueous humor outflow resistance to restore IOP to acceptable levels.

Extracellular matrix turnover and outflow resistance.

Normal homeostatic adjustment of elevated intraocular pressure (IOP) involves remodeling the extracellular matrix (ECM) of the trabecular meshwork (TM). This entails sensing elevated IOP, releasing numerous activated proteinases to degrade existing ECM and concurrent biosynthesis of replacement ECM components. To increase or decrease IOP, the quantity, physical properties and/or organization of new components should be somewhat different from those replaced in order to modify outflow resistance.

Specialized podosome- or invadopodia-like structures (PILS) for focal trabecular meshwork extracellular matrix turnover.

Purpose: There are distinctive areas of colocalization of matrix metalloproteinase (MMP)-2 and -14 on trabecular meshwork (TM) cells that resemble podosomes or invadopodia. Studies were conducted to determine whether TM cells exhibit podosome- or invadopodia-like structures (PILS) and whether they produce focal extracellular matrix (ECM) turnover.

Methods: Porcine and human TM cells and perfused anterior segment organ cultures were studied.

Nucleotide receptor signaling in murine macrophages is linked to reactive oxygen species generation.

Macrophage activation is critical in the innate immune response and can be regulated by the nucleotide receptor P2X7. In this regard, P2X7 signaling is not well understood but has been implicated in controlling reactive oxygen species (ROS) generation by various leukocytes. Although ROS can contribute to microbial killing, the role of ROS in nucleotide-mediated cell signaling is unclear.

The nucleotide receptor P2X7 mediates actin reorganization and membrane blebbing in RAW 264.7 macrophages via p38 MAP kinase and Rho.

Extracellular nucleotides regulate macrophage function via P2X nucleotide receptors that form ligand-gated ion channels. In particular, P2X7 activation is characterized by pore formation, membrane blebbing, and cytokine release. P2X7 is also linked to mitogen-activated protein kinases (MAPK) and Rho-dependent pathways, which are known to affect cytoskeletal structure in other systems.

Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-kappa B signaling pathways in murine RAW 264.7 macrophages.

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X(7). In this regard, after exposure to bacterial LPS, P2X(7) activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X(7) has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-kappa B, we tested the hypothesis that LPS and nucleotides regulate NF-kappa B-dependent inflammatory events via cross talk with MAPK-associated pathways.

Purinergic receptor regulation of LPS-induced signaling and pathophysiology.

Macrophages express several lipopolysaccharide (LPS) binding proteins and are potently activated by LPS to produce inflammatory mediators. Recent studies have shown that receptors for exogenous nucleotides (P2X and P2Y purinergic receptors) can modulate macrophage production of TNF-alpha, IL-1beta and nitric oxide (NO) following LPS exposure. Macrophages and LPS-stimulated monocytes express elevated levels of P2Y1, P2Y2 and P2X7 mRNA, suggesting that both P2Y and P2X receptors can contribute to LPS-induced pathophysiology.

Modulation of monocyte signaling and pore formation in response to agonists of the nucleotide receptor P2X(7).

Previous reports about the nucleotide receptor P2X(7), which exhibits ion channel and pore-forming activity and is known to promote IL-1beta processing, have centered largely on its role in macrophage function, whereas its participation in monocyte activity has been unclear. However, because extracellular ATP has been shown to affect monocytes with respect to IL-1beta release, we hypothesized that the P2X(7) receptor is also present and functional in a subpopulation of blood monocytes. Flow cytometric analysis revealed that about 70% of monocytes isolated from normal human donors expressed the P2X(7) receptor.