A. Thomas Read

Role of nitric oxide in murine conventional outflow physiology

Elevated intraocular pressure (IOP) is the main risk factor for glaucoma. Exogenous nitric oxide (NO) decreases IOP by increasing outflow facility, but whether endogenous NO production contributes to the physiological regulation of outflow facility is unclear. Outflow facility was measured by pressure-controlled perfusion in ex vivo eyes from C57BL/6 wild-type (WT) or transgenic mice expressing human endothelial NO synthase (eNOS) fused to green fluorescent protein (GFP) superimposed on the endogenously expressed murine eNOS (eNOS-GFPtg). In WT mice, exogenous NO delivered by 100 μM S-nitroso-N-acetylpenicillamine (SNAP) increased outflow facility by 62 ± 28% (SD) relative to control eyes perfused with the inactive SNAP analog N-acetyl-d-penicillamine (NAP; n = 5, P = 0.016). In contrast, in eyes from eNOS-GFPtg mice, SNAP had no effect on outflow facility relative to NAP (-9 ± 4%, P = 0.40). In WT mice, the nonselective NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME, 10 μM) decreased outflow facility by 36 ± 13% (n = 5 each, P = 0.012), but 100 μM l-NAME had no detectable effect on outflow facility (-16 ± 5%, P = 0.22). An eNOS-selective inhibitor (cavtratin, 50 μM) decreased outflow facility by 19 ± 12% in WT (P = 0.011) and 39 ± 25% in eNOS-GFPtg (P = 0.014) mice. In the conventional outflow pathway of eNOS-GFPtg mice, eNOS-GFP expression was localized to endothelial cells lining Schlemms canal and the downstream vessels, with no apparent expression in the trabecular meshwork. These results suggest that endogenous NO production by eNOS within endothelial cells of Schlemms canal or downstream vessels contributes to the physiological regulation of aqueous humor outflow facility in mice, representing a viable strategy to more successfully lower IOP in glaucoma.

Young’s modulus of elasticity of Schlemm’s canal endothelial cells

Schlemm’s canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young’s modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young’s modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8–15 mm Hg). However, increasing intraocular pressure (22–30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.

Sphingosine-1-phosphate effects on the inner wall of Schlemm's canal and outflow facility in perfused human eyes

Previous work has shown that sphingosine 1-phosphate (S1P) decreases outflow facility in perfused porcine eyes while dramatically increasing giant vacuole density in the inner wall of the aqueous plexus, with no obvious changes in the trabecular meshwork (TM). Due to known effects of S1P on cell-cell junction assembly in vascular endothelia, we hypothesized that S1P would decrease outflow facility in human eyes by increasing the complexity of cell-cell junctions in Schlemms canal (SC) inner wall endothelia. Perfusion of enucleated post mortem human eyes at 8 mmHg constant pressure in the presence or absence of 5 microM S1P showed that S1P decreased outflow facility by 36 +/- 20% (n = 10 pairs; p = 0.0004); an effect likely mediated by activation of S1P(1) and/or S1P(3) receptor subtypes, which were found to be the principal S1P receptors expressed by both TM and SC cells by RT-PCR, confocal immunofluorescence microscopy and western blot analyses. Examination of SCs inner wall using confocal microscopy revealed no consistent differences in VE-cadherin, beta-catenin, phosphotyrosine or filamentous actin abundance/distribution between S1P-treated eyes and controls. Moreover, morphological inspection of conventional outflow tissues by light and scanning electron microscopy showed no significant differences between S1P-treated and control eyes, particularly in giant vacuole density. Thus, unlike the situation in porcine eyes, we did not observe changes in inner wall morphology in human eyes treated with S1P, despite a significant and immediate decrease in outflow facility in both species. Regardless, S1P receptor antagonists represent novel therapeutic prospects for ocular hypertension in humans.

In vitro models for glaucoma research: Effects of hydrostatic pressure

PURPOSE The response of cells (e.g., optic nerve head [ONH] cells) to mechanical stress is important in glaucoma. Studies have reported the biological effects of hydrostatic pressure on ONH cells cultured on a rigid substrate. An apparatus, designed to independently vary hydrostatic pressure and gas tension (including oxygen tension) in culture medium, was used to evaluate the effects of pressure and tension on cell migration, shape, and α-tubulin architecture in a transformed cell line (DITNC1 rat cortical astrocytes). METHODS During the assay period, cells were exposed to one of four experimental configurations: (1) control pressure and control gas tension; (2) high-pressure (7.4 mm Hg) and reduced gas tension; (3) control pressure and reduced gas tension; and (4) high-pressure and control gas tension. RESULTS Calculations suggested that the cells in configurations 2 and 3 were hypoxic, as confirmed by direct measurements in configuration 2. No effects of hydrostatic pressure were observed on cell migration or α-tubulin architecture. However, cells cultured under low gas tension (configurations 2 and 3) showed increased migration at 48 and 72 hours (P < 0.05). CONCLUSIONS A hydrostatic pressure of 7.4 mm Hg has no effect on DITNC1 astrocytes cultured on rigid coverslips, whereas hypoxia associated with a fluid column creating this pressure does. These results differ from those in a previous report, the results of which may be explained by altered gas tensions in the culture medium. Steps are recommended for control of secondary effects when testing the effect of pressure on cultured cells.

Trabecular meshwork stiffness in glaucoma

ABSTRACT Alterations in stiffness of the trabecular meshwork (TM) may play an important role in primary open‐angle glaucoma (POAG), the second leading cause of blindness. Specifically, certain data suggest an association between elevated intraocular pressure (IOP) and increased TM stiffness; however, the underlying link between TM stiffness and IOP remains unclear and requires further study. We here first review the literature on TM stiffness measurements, encompassing various species and based on a number of measurement techniques, including direct approaches such as atomic force microscopy (AFM) and uniaxial tension tests, and indirect methods based on a beam deflection model. We also briefly review the effects of several factors that affect TM stiffness, including lysophospholipids, rho‐kinase inhibitors, cytoskeletal disrupting agents, dexamethasone (DEX), transforming growth factor‐Symbol (TGF‐Symbol), nitric oxide (NO) and cellular senescence. We then describe a method we have developed for determining TM stiffness measurement in mice using a cryosection/AFM‐based approach, and present preliminary data on TM stiffness in C57BL/6J and CBA/J mouse strains. Finally, we investigate the relationship between TM stiffness and outflow facility between these two strains. The method we have developed shows promise for further direct measurements of mouse TM stiffness, which may be of value in understanding mechanistic relations between outflow facility and TM biomechanical properties. Symbol. No caption available. Symbol. No caption available. HIGHLIGHTSTrabecular meshwork stiffness may be an important contributor to raised IOP.Measurements of trabecular meshwork (TM) stiffness, and factors affecting this quantity, are reviewed.A new technique to directly measure TM stiffness in mouse eyes is presented.Preliminary data relating TM stiffness and outflow facility in C57BL/6J and CBA/J mice are shown.

A New Method for Selection of Angular Aqueous Plexus Cells from Porcine Eyes: A Model for Schlemm's Canal Endothelium

PURPOSE The authors sought to develop a technique for isolating and culturing angular aqueous plexus (AAP) cells from more plentiful porcine eyes. AAP is an analogue of Schlemms canal. METHODS Cells were differentially selected with puromycin, a toxin often used to select brain microvascular endothelial cells based on the expression of P-glycoprotein (P-gp), a multidrug resistance efflux pump. Trabecular meshwork containing AAP was dissected and pooled from fresh porcine eyes, digested in collagenase I, washed, filtered, and cultured for 8 days in a gelatin-coated plastic flask. Cells were then selected by exposure to 4 μg/mL puromycin for 2 days in the culture medium. Cells were fixed and immunostained for P-gp, ICAM II, von Willebrand factor (vWF), VE-cadherin, and α-smooth muscle actin (α-SMA). RESULTS Histology of the limbus showed that the dissection was limited to the trabecular meshwork region, including the AAP. Before puromycin treatment, cells appeared heterogeneous and polygonal, suggestive of a mixed population. More than 90% of the cells were removed by puromycin, leaving a population that appeared uniformly cobblestone-like when grown to confluence and that was contact inhibited. Puromycin-selected cells stained positively for the endothelial markers ICAM II, vWF, and VE-cadherin but negatively for α-SMA, consistent with staining patterns in whole tissue. CONCLUSIONS Based on marker expression, morphology, and behavior in culture, puromycin-selected cells from porcine outflow tissues are AAP endothelial cells. Thus, porcine eyes can provide a plentiful alternative cell source for studying Schlemms canal biology related to ocular hypertension.

The role of the calceoli in precopulatory behaviour and mate recognition of Gammarus pseudolimnaeus Bousfield (Crustacea, Amphipoda)

The importance of calceoli (male antennular organs) in mate recognition of Gammarus pseudolimnaeus Bousfield was examined in the laboratory. The relationship between female receptivity and their moult stage was examined by comparing the moult stage of amplexed and non-amplexed females. The former were clearly in later moult stages and thus closer to ovulation. Ablation of the antennular 2 flagella, which uniquely possess the calceoli, had no effect on the ability of males to find, evaluate and amplex with receptive females. Morphological examination verified the complex nature and apparent lack of innervation of calceoli. We propose that the calceolus functions as a general environmental vibrioreceptor, necessary in the male because of its greater level of activity.

Enhancement of Outflow Facility in the Murine Eye by Targeting Selected Tight-Junctions of Schlemm's Canal Endothelia.

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm’s canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system.

Quantification of the efficacy of collagen cross-linking agents to induce stiffening of rat sclera

The concept of scleral stiffening therapies has emerged as a novel theoretical approach for treating the ocular disorders glaucoma and myopia. Deformation of specific regions of the posterior eye is innately involved in the pathophysiology of these diseases, and thus targeted scleral stiffening could resist these changes and slow or prevent progression of these diseases. Here, we present the first systematic screen and direct comparison of the stiffening effect of small molecule collagen cross-linking agents in the posterior globe, namely using glyceraldehyde, genipin and methylglyoxal (also called pyruvaldehyde). To establish a dose–response relationship, we used inflation testing to simulate the effects of increasing intraocular pressure in freshly harvested rat eyes stiffened with multiple concentrations of each agent. We used digital image correlation to compute the mechanical strain in the tissue as a metric of stiffness, using a novel treatment paradigm for screening relative stiffening by incubating half of each eye in cross-linker and using the opposite half as an internal control. We identified the doses necessary to increase stiffness by approximately 100%, namely 30 mM for glyceraldehyde, 1 mM for genipin and 7 mM for methylglyoxal, and we also identified the range of stiffening it was possible to achieve with such agents. Such findings will inform development of in vivo studies of scleral stiffening to treat glaucoma and myopia.

The relationship between outflow resistance and trabecular meshwork stiffness in mice

It has been suggested that common mechanisms may underlie the pathogenesis of primary open-angle glaucoma (POAG) and steroid-induced glaucoma (SIG). The biomechanical properties (stiffness) of the trabecular meshwork (TM) have been shown to differ between POAG patients and unaffected individuals. While features such as ocular hypertension and increased outflow resistance in POAG and SIG have been replicated in mouse models, whether changes of TM stiffness contributes to altered IOP homeostasis remains unknown. We found that outer TM was stiffer than the inner TM and, there was a significant positive correlation between outflow resistance and TM stiffness in mice where conditions are well controlled. This suggests that TM stiffness is intimately involved in establishing outflow resistance, motivating further studies to investigate factors underlying TM biomechanical property regulation. Such factors may play a role in the pathophysiology of ocular hypertension. Additionally, this finding may imply that manipulating TM may be a promising approach to restore normal outflow dynamics in glaucoma. Further, novel technologies are being developed to measure ocular tissue stiffness in situ. Thus, the changes of TM stiffness might be a surrogate marker to help in diagnosing altered conventional outflow pathway function if those technologies could be adapted to TM.