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.

Neuropsin, TRPV4 and intracellular calcium mediate intrinsic photosensitivity in corneal epithelial cells.

Purpose: To investigate intrinsic phototransduction in the corneal epithelium and its role in intracellular and inflammatory signaling.

Methods: Optical imaging in isolated corneal epithelial cells (CECs) and debrided epithelia was combined with molecular, biochemical, pharmacological assays and gene deletion studies to track UVB-induced calcium signaling and release of cytokines, chemokines and matrix remodeling enzymes. Results from wild type mouse CECs were compared to data obtained from Opn5 and Trpv4 cells.

TRPV4 overactivation enhances cellular contractility and drives ocular hypertension in TGFβ2 overexpressing eyes.

The risk for developing primary open-angle glaucoma (POAG) correlates with the magnitude of ocular hypertension (OHT) and the concentration of transforming growth factor-β2 (TGFβ2) in the aqueous humor. Effective treatment of POAG requires detailed understanding of interaction between pressure sensing mechanisms in the trabecular meshwork (TM) and biochemical risk factors. Here, we employed molecular, optical, electrophysiological and tonometric strategies to establish the role of TGFβ2 in transcription and functional expression of mechanosensitive channel isoforms alongside studies of TM contractility in biomimetic hydrogels, and intraocular pressure (IOP) regulation in a mouse model of TGFβ2 -induced OHT.

Resting human trabecular meshwork cells experience tonic cation influx.

The trabecular meshwork (TM) regulates intraocular pressure (IOP) by converting biochemical and biomechanical stimuli into intracellular signals. Recent electrophysiological studies demonstrated that this process is mediated by pressure sensing ion channels in the TM plasma membrane while the molecular and functional properties of channels that underpin ionic homeostasis in resting cells remain largely unknown. Here, we demonstrate that the TM resting potential is subserved by a powerful cationic conductance that disappears following Na removal and substitution with choline or NMDG.

TRPV4 subserves physiological and pathological elevations in intraocular pressure.

Ocular hypertension (OHT) caused by mechanical stress and chronic glucocorticoid exposure reduces the hydraulic permeability of the conventional outflow pathway. It increases the risk for irreversible vision loss, yet healthy individuals experience nightly intraocular pressure (IOP) elevations without adverse lifetime effects. It is not known which pressure sensors regulate physiological vs.

TRPV4 and chloride channels mediate volume sensing in trabecular meshwork cells.

Aqueous humor drainage from the anterior eye determines intraocular pressure (IOP) under homeostatic and pathological conditions. Swelling of the trabecular meshwork (TM) alters its flow resistance but the mechanisms that sense and transduce osmotic gradients remain poorly understood. We investigated TM osmotransduction and its role in calcium and chloride homeostasis using molecular analyses, optical imaging, and electrophysiology.

Regulation of Corneal Stromal Cell Behavior by Modulating Curvature Using a Hydraulically Controlled Organ Chip Array.

Curvature is a critical factor in cornea mechanobiology, but its impact on phenotypic alterations and extracellular matrix remodeling of cornea stroma remains unclear. In this work, we investigated how curvature influences the corneal stroma using a hydraulically controlled curvature array chip. The responses of stromal cells to low, medium, and high curvatures were observed by preparing three phenotypes of corneal stromal cells: corneal keratocytes, fibroblasts, and myofibroblasts.

TRPV4: Cell type-specific activation, regulation and function in the vertebrate eye.

The architecture of the vertebrate eye is optimized for efficient delivery and transduction of photons and processing of signaling cascades downstream from phototransduction. The cornea, lens, retina, vasculature, ciliary body, ciliary muscle, iris and sclera have specialized functions in ocular protection, transparency, accommodation, fluid regulation, metabolism and inflammatory signaling, which are required to enable function of the retina-light sensitive tissue in the posterior eye that transmits visual signals to relay centers in the midbrain. This process can be profoundly impacted by non-visual stimuli such as mechanical (tension, compression, shear), thermal, nociceptive, immune and chemical stimuli, which target these eye regions to induce pain and precipitate vision loss in glaucoma, diabetic retinopathy, retinal dystrophies, retinal detachment, cataract, corneal dysfunction, ocular trauma and dry eye disease.

Retinal TRP channels: Cell-type-specific regulators of retinal homeostasis and multimodal integration.

Transient receptor potential (TRP) channels are a widely expressed family of 28 evolutionarily conserved cationic ion channels that operate as primary detectors of chemical and physical stimuli and secondary effectors of metabotropic and ionotropic receptors. In vertebrates, the channels are grouped into six related families: TRPC, TRPV, TRPM, TRPA, TRPML, and TRPP. As sensory transducers, TRP channels are ubiquitously expressed across the body and the CNS, mediating critical functions in mechanosensation, nociception, chemosensing, thermosensing, and phototransduction.

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.

TRPV4 and TRPC1 channels mediate the response to tensile strain in mouse Müller cells.

Müller glia, a pillar of metabolic, volume regulatory and immune/inflammatory signaling in the mammalian retina, are among the earliest responders to mechanical stressors in the eye. Ocular trauma, edema, detachment and glaucoma evoke early inflammatory activation of Müller cells yet the identity of their mechanotransducers and signaling mechanisms downstream remains unknown. Here, we investigate expression of genes that encode putative stretch-activated calcium channels (SACs) in mouse Müller cells and study their responses to dynamical tensile loading in cells loaded with a calcium indicator dye.

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.

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression, and the cellular response to tension.

Despite the association of cholesterol with debilitating pressure-related diseases such as glaucoma, heart disease, and diabetes, its role in mechanotransduction is not well understood. We investigated the relationship between mechanical strain, free membrane cholesterol, actin cytoskeleton, and the stretch-activated transient receptor potential vanilloid isoform 4 (TRPV4) channel in human trabecular meshwork (TM) cells. Physiological levels of cyclic stretch resulted in time-dependent decreases in membrane cholesterol/phosphatidylcholine ratio and upregulation of stress fibers.

The RNA-binding protein and stress granule component ATAXIN-2 is expressed in mouse and human tissues associated with glaucoma pathogenesis.

Polyglutamine repeat expansions in the Ataxin-2 (ATXN2) gene were first implicated in Spinocerebellar Ataxia Type 2, a disease associated with degeneration of motor neurons and Purkinje cells. Recent studies linked single nucleotide polymorphisms in the gene to elevated intraocular pressure in primary open angle glaucoma (POAG); yet, the localization of ATXN2 across glaucoma-relevant tissues of the vertebrate eye has not been thoroughly examined. This study characterizes ATXN2 expression in the mouse and human retina, and anterior eye, using an antibody validated in ATXN2 retinas.

TRPV4-Rho signaling drives cytoskeletal and focal adhesion remodeling in trabecular meshwork cells.

Intraocular pressure (IOP) is dynamically regulated by the trabecular meshwork (TM), a mechanosensitive tissue that protects the eye from injury through dynamic regulation of aqueous humor flow. TM compensates for mechanical stress impelled by chronic IOP elevations through increased actin polymerization, tissue stiffness, and contractility. This process has been associated with open angle glaucoma; however, the mechanisms that link mechanical stress to pathological cytoskeletal remodeling downstream from the mechanotransducers remain poorly understood.

TRPV4 channels mediate the mechanoresponse in retinal microglia.

The physiological and neurological correlates of plummeting brain osmolality during edema, traumatic CNS injury, and severe ischemia are compounded by neuroinflammation. Using multiple approaches, we investigated how retinal microglia respond to challenges mediated by increases in strain, osmotic gradients, and agonists of the stretch-activated cation channel TRPV4. Dissociated and intact microglia were TRPV4-immunoreactive and responded to the selective agonist GSK1016790A and substrate stretch with altered motility and elevations in intracellular calcium ([Ca ] ).

Piezo1 channels mediate trabecular meshwork mechanotransduction and promote aqueous fluid outflow.

Key Points: Trabecular meshwork (TM) is a highly mechanosensitive tissue in the eye that regulates intraocular pressure through the control of aqueous humour drainage. Its dysfunction underlies the progression of glaucoma but neither the mechanisms through which TM cells sense pressure nor their role in aqueous humour outflow are understood at the molecular level. We identified the Piezo1 channel as a key TM transducer of tensile stretch, shear flow and pressure.

Magneto-mechanical actuation of barium-hexaferrite nanoplatelets for the disruption of phospholipid membranes.

Hypothesis: The magneto-mechanical actuation (MMA) of magnetic nanoparticles with a low-frequency alternating magnetic field (AMF) can be used to destroy cancer cells. So far, MMA was tested on different cells using different nanoparticles and different field characteristics, which makes comparisons and any generalizations about the results of MMA difficult. In this paper we propose the use of giant unilamellar vesicles (GUVs) as a simple model system to study the effect of MMA on a closed lipid bilayer membrane, i.

TRPV1 activation stimulates NKCC1 and increases hydrostatic pressure in the mouse lens.

The porcine lens response to a hyperosmotic stimulus involves an increase in the activity of an ion cotransporter sodium-potassium/two-chloride cotransporter 1 (NKCC1). Recent studies with agonists and antagonists pointed to a mechanism that appears to depend on activation of transient receptor potential vanilloid 1 (TRPV1) ion channels. Here, we compare responses in lenses and cultured lens epithelium obtained from TRPV1 and wild type (WT) mice.

Polymodal Sensory Transduction in Mouse Corneal Epithelial Cells.

Purpose: Contact lenses, osmotic stressors, and chemical burns may trigger severe discomfort and vision loss by damaging the cornea, but the signaling mechanisms used by corneal epithelial cells (CECs) to sense extrinsic stressors are not well understood. We therefore investigated the mechanisms of swelling, temperature, strain, and chemical transduction in mouse CECs.

Methods: Intracellular calcium imaging in conjunction with electrophysiology, pharmacology, transcript analysis, immunohistochemistry, and bioluminescence assays of adenosine triphosphate (ATP) release were used to track mechanotransduction in dissociated CECs and epithelial sheets isolated from the mouse cornea.

Volume sensing in the transient receptor potential vanilloid 4 ion channel is cell type-specific and mediated by an N-terminal volume-sensing domain.

Many retinal diseases are associated with pathological cell swelling, but the underlying etiology remains to be established. A key component of the volume-sensitive machinery, the transient receptor potential vanilloid 4 (TRPV4) ion channel, may represent a sensor and transducer of cell swelling, but the molecular link between the swelling and TRPV4 activation is unresolved. Here, our results from experiments using electrophysiology, cell volumetric measurements, and fluorescence imaging conducted in murine retinal cells and oocytes indicated that cell swelling in the physiological range activated TRPV4 in Müller glia and oocytes, but required phospholipase A (PLA) activity exclusively in Müller cells.