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.

The session centered around three questions: What is the evidence that the pathophysiology of ocular hypertension is cell mediated, how do outflow cells deal with stress, and how does the aqueous humor enter Schlemm's canal? The discussion revealed several areas in which research could aid in our

Polymorphisms in the CAV1/2 genes that encode signature proteins of caveolae are associated with glaucoma, the second leading cause of blindness worldwide, and with its major risk factor, intraocular pressure (IOP). We hypothesized that caveolin-1 (Cav-1) participates in IOP maintenance via modulation of aqueous humor drainage from the eye. We localize caveolae proteins to human and murine conventional drainage tissues and show that caveolae respond to mechanical stimulation. We show that Cav-1-deficient (Cav-1−/−) mice display ocular hypertension explained by reduced pressure-dependent drainage of aqueous humor. Cav-1 deficiency results in loss of caveolae in the Schlemm’s canal (SC) and trabecular meshwork. However, their absence did not appear to impact development nor adult form of the conventional outflow tissues according to rigorous quantitative ultrastructural analyses, but did affect cell and tissue behavior. Thus, when IOP is experimentally elevated, cells of the Cav-1−/− outflow tissues are more susceptible to plasma membrane rupture indicating that caveolae play a role in mechanoprotection. Additionally, aqueous drainage from Cav-1−/− eyes was more sensitive to nitric oxide (NO) synthase inhibition than controls, suggesting that excess NO partially compensates for outflow pathway dysfunction. These results provide a functional link between a glaucoma risk gene and glaucoma-relevant pathophysiology.

The cause of the elevated outflow resistance and consequent ocular hypertension characteristic of glaucoma is unknown. To investigate possible causes for this flow resistance, we used atomic force microscopy (AFM) with 10-µm spherical tips to probe the stiffness of the inner wall of Schlemm’s canal as a function of distance from the tissue surface in normal and glaucomatous postmortem human eyes, and 1-µm spherical AFM tips to probe the region immediately below the tissue surface. To localize flow resistance, perfusion and imaging methods were used to characterize the pressure drop in the immediate vicinity of the inner wall using giant vacuoles that form in Schlemm’s canal cells as micropressure sensors. Tissue stiffness increased with increasing AFM indentation depth. Tissues from glaucomatous eyes were stiffer compared with normal eyes, with greatly increased stiffness residing within ∼1 µm of the inner-wall surface. Giant vacuole size and density were similar in normal and glaucomatous eyes despite lower flow rate through the latter due to their higher flow resistance. This implied that the elevated flow resistance found in the glaucomatous eyes was localized to the same region as the increased tissue stiffness. Our findings implicate pathological changes to biophysical characteristics of Schlemm’s canal endothelia and/or their immediate underlying extracellular matrix as cause for ocular hypertension in glaucoma.

SUMMARYThe age-related changes of the ciliary muscle of human eyes (33-87 years) were studied on histological meridional sections. Eighty-five melanoma eyes and 10 eyes of normal donors were investigated. The total area and the length of the muscle, the area of the three main portions and the distance of the inner apex of the muscle to the scleral spur were determined and correlated with age. Total area and length of the muscle show a continuous and significant decrease with age. The area of the longitudinal and reticular portion continuously decreases, whereas the area of the circular portion significantly increases with age. The decrease in area is more pronounced in the longitudinal portion than in the reticular portion of the muscle, which shows an age-related increase in connective tissue. In addition, the distance of the inner apex of the muscle to the scleral spur shortens continuously. Thus, with increasing age the ciliary muscle adopts an anterior-inward position. A similar form is seen in young eyes after ciliary muscle contraction only. There might be a functional relationship between the observed age-changes in the ciliary muscle system and the phenomenon of the so-called qens paradox' (steepening of the anterior and posterior curvatures of the disaccommodated lens with age).

The biomechanical environment within the optic nerve head (ONH) is complex and is likely directly involved in the loss of retinal ganglion cells (RGCs) in glaucoma. Unfortunately, our understanding of this process is poor. Here we describe factors that influence ONH biomechanics, including ONH connective tissue microarchitecture and anatomy; intraocular pressure (IOP); and cerebrospinal fluid pressure (CSFp). We note that connective tissue factors can vary significantly from one individual to the next, as well as regionally within an eye, and that the understanding of ONH biomechanics is hindered by anatomical differences between small-animal models of glaucoma (rats and mice) and humans. Other challenges of using animal models of glaucoma to study the role of biomechanics include the complexity of assessing the degree of glaucomatous progression; and inadequate tools for monitoring and consistently elevating IOP in animal models. We conclude with a consideration of important open research questions/challenges in this area, including: (i) Creating a systems biology description of the ONH; (ii) addressing the role of astrocyte connective tissue remodeling and reactivity in glaucoma; (iii) providing a better characterization of ONH astrocytes and non-astrocytic constituent cells; (iv) better understanding the role of ONH astrocyte phagocytosis, proliferation and death; (v) collecting gene expression and phenotype data on a larger, more coordinated scale; and (vi) developing an implantable IOP sensor.

Differentiating oligodendrocytes generate myelin to ensure rapid saltatory conduction in the vertebrate central nervous system. Although oligodendroglial differentiation and myelination are accompanied by dramatic chromatin reorganizations, previously studied chromatin remodelers had only limited direct effects on the process. To study the functional significance of chromatin changes for myelination and identify relevant remodelers, we deleted Ep400, the central ATP-hydrolyzing subunit of the TIP60/EP400 complex, at defined times of mouse oligodendrocyte development. Whereas Ep400-deficient oligodendrocyte precursors develop normally, terminal differentiation and myelination are dramatically impaired. Mechanistically, Ep400 interacts with transcription factor Sox10, binds to regulatory regions of the Myrf gene and is required to induce this central transcriptional regulator of the myelination program. In addition to reduced and aberrant myelin formation, oligodendrocytes exhibit increased DNA damage and apoptosis so that numbers never reach wildtype levels during the short lifespan of Ep400-deficient mice. Ep400 deletion in already mature oligodendrocytes remains phenotypically inapparent arguing that Ep400 is dispensable for myelin maintenance. Given its essential function in myelin formation, modulation of Ep400 activity may be beneficial in conditions such as multiple sclerosis where this process is compromised.

The molecular pathogenesis of choroidal neovascularization (CNV), an angiogenic process that critically contributes to vision loss in age-related macular degeneration, is unclear. Herein, we analyzed the role of transforming growth factor (TGF)-β signaling for CNV formation by generating a series of mutant mouse models with induced conditional deletion of TGF-β signaling in the entire eye, the retinal pigment epithelium (RPE), or the vascular endothelium. Deletion of TGF-β signaling in the eye caused CNV, irrespectively if it was ablated in newborn or 3-week-old mice. Areas of CNV showed photoreceptor degeneration, multilayered RPE, basal lamina deposits, and accumulations of monocytes/macrophages. The changes progressed, leading to marked structural and functional alterations of the retina. Although the specific deletion of TGF-β signaling in the RPE caused no obvious changes, specific deletion in vascular endothelial cells caused CNV and a phenotype similar to that observed after the deletion in the entire eye. We conclude that impairment of TGF-β signaling in the vascular endothelium of the eye is sufficient to trigger CNV formation. Our findings highlight the importance of TGF-β signaling as a key player in the development of ocular neovascularization and indicate a fundamental role of TGF-β signaling in the pathogenesis of age-related macular degeneration.