Colocalization of outflow segmentation and pores along the inner wall of Schlemm's canal.

All aqueous humor draining through the conventional outflow pathway must cross the endothelium of Schlemm's canal (SC), likely by passing through micron-sized transendothelial pores. SC pores are non-uniformly distributed along the inner wall endothelium, but it is unclear how the distribution of pores relates to the non-uniform or segmental distribution of aqueous humor outflow through the trabecular meshwork. It is hypothesized that regions in the juxtacanalicular tissue (JCT) with higher local outflow should coincide with regions of greater inner wall pore density compared to JCT regions with lower outflow. Three pairs of non-glaucomatous human donor eyes were perfused at 8 mmHg with fluorescent tracer nanospheres to decorate local patterns of outflow segmentation through the JCT. The inner wall was stained for CD31 and/or vimentin and imaged en face using confocal and scanning electron microscopy (SEM). Confocal and SEM images were spatially registered to examine the spatial relationship between inner wall pore density and tracer intensity in the underlying JCT. For each eye, tracer intensity, pore density (n) and pore diameter (D) (for both transcellular "I" and paracellular "B" pores) were measured in 4-7 regions of interest (ROIs; 50 × 150 µm each). Analysis of covariance was used to examine the relationship between tracer intensity and pore density, as well as the relationship between tracer intensity and three pore metrics (nD, nD(2) and nD(3)) that represent the local hydraulic conductivity of the outflow pathway as predicted by various hydrodynamic models. Tracer intensity in the JCT correlated positively with local pore density when considering total pores (p = 0.044) and paracellular B pores on their own (p = 0.016), but not transcellular I-pores on their own (p = 0.54). Local hydraulic conductivity as predicted by the three hydrodynamic models all showed a significant positive correlation with tracer intensity when considering total pores and B-pores (p < 0.0015 and p < 10(-4)) but not I-pores (p > 0.38). These data suggest that aqueous humor passes through micron-sized pores in the inner wall endothelium of SC. Paracellular B-pores appear to have a dominant contribution towards transendothelial filtration across the inner wall relative to transcellular I-pores. Impaired pore formation, as previously described in glaucomatous SC cells, may thereby contribute to greater outflow heterogeneity, outflow obstruction, and IOP elevation 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.

Aquaporin-1 expression and conventional aqueous outflow in human eyes.

Aquaporin channels facilitate the enhanced permeability of secretory and absorptive tissues to water. In the conventional drainage tract, aquaporin-1 is expressed but its contribution to outflow facility is unknown. The purpose of the present study was to determine the effect of elevated aquaporin-1 expression by cells of the human conventional drainage pathway on outflow facility. Using 13 pairs of human anterior segments in organ culture, we modified aquaporin-1 protein expression in outflow cells using adenovirus encoding human aquaporin-1. Contralateral anterior segments served as controls and were transduced with adenovirus encoding beta-galactosidase. By confocal immunofluorescence microscopy, we observed that inner trabecular meshwork cells from anterior segments exposed to adenovirus (via injection into the inlet tubing during perfusion) had increased aquaporin-1 protein expression compared to endogenous levels. In contrast, elevation of aquaporin-1 protein in outer meshwork cells (juxtacanalicular region) and Schlemm's canal required transduction of adenovirus into anterior segments using retroperfusion via episcleral veins. Regardless of exposure route, outflow facility of experimental segments was not different than control. Specifically, overexpression of aquaporin-1 in the inner meshwork resulted in an average facility change of -2.0+/-9.2%, while overexpression of aquaporin-1 in the resistance-generating region changed outflow facility by -3.2+/-11.2%. Taken together, these results indicate that a transcellular pathway, mediated by aquaporin-1, does not contribute significantly to bulk outflow through the conventional aqueous outflow tract of human eyes.

Actin structure in the outflow tract of normal and glaucomatous eyes.

To characterize the in situ distribution of actin in Schlemm's canal endothelium (SCE) and juxtacanalicular tissue (JCT) cells in glaucomatous human eyes, and compare to the distribution in normal eyes. Fresh human eye bank eyes were perfused and fixed at pressure (n=27 normal eyes and 22 confirmed glaucomatous eyes). Schlemm's canal was opened by microdissection and outflow tissues were labelled for confocal microscopy to visualize F-actin, nuclei, laminin and/or CD31. Images were acquired in Z-series from the inner wall of Schlemm's canal, juxtacanalicular tissue and outer corneoscleral meshwork. In normal eyes, inner wall Schlemm's canal endothelial (SCE) cells showed a dense peripheral F-actin band, as previously described. JCT cells showed a more random and amorphous F-actin distribution. In glaucoma eyes, peripheral F-actin bands were less common in inner wall SCE cells; instead, F-actin was more centrally located within the cell and appeared "tangled". These actin tangles were also prominent in JCT cells of glaucoma eyes. Glaucoma eyes also demonstrated structures with features of cross-linked actin networks (CLANs), and more frequent occurrence of punctuate actin concentrations. There was a significant degree of heterogeneity, with some regions from glaucomatous eyes appearing normal and vice versa. F-actin architecture in human outflow pathway cells in situ differs between normal and glaucoma eyes, with glaucomatous tissue showing a more "disordered" actin architecture overall. Some of these changes are likely due to effects secondary to administration of anti-glaucoma medications. Most of the changes that we observed could potentially affect the biomechanical properties of the outflow pathway tissues in glaucoma, but their role in the pathogenesis of ocular hypertension remains unclear.

Effects of latrunculin-B on outflow facility and trabecular meshwork structure in human eyes.

PURPOSE: To determine the effect of the F-actin-disrupting agent latrunculin-B on aqueous outflow facility and trabecular meshwork architecture in human eyes. METHODS: After baseline facility measurement in human eye bank eyes (n = 9 pairs), one eye of each pair received anterior chamber exchange and continued perfusion with medium containing 1 microM latrunculin-B. Contralateral eyes were treated in a similar manner with vehicle. Eyes were fixed by anterior chamber exchange and perfusion with universal fixative at 8 mm Hg (corresponding to a physiologic pressure of 15 mm Hg in vivo), and outflow pathway tissues were examined by transmission and scanning electron microscopy. RESULTS: Perfusion of eyes with 1 microM latrunculin-B caused a continuous and ongoing increase in outflow facility, resulting in a net facility difference of 64% 2 hours after drug administration (P < 0.006). Transmission electron microscopy showed subtle and focal detachment of the inner wall of Schlemm's canal, rarefaction of the juxtacanalicular tissue (JCT), and cell-cell and cell-matrix detachment. Scanning electron microscopy showed collapsed vacuoles in the inner wall of Schlemm's canal and a marked increase in the number and size of border (paracellular) pores in the inner wall. CONCLUSIONS: Latrunculin-B increases outflow facility in postmortem human eyes. The mechanism of facility increase is most likely due to loss of mechanical integrity of the trabecular meshwork as a consequence of reduction in cell-cell and cell-matrix adhesion. The facility increase and the extent of inner wall separation from the JCT that we observed were both qualitatively similar to that reported in living monkey eyes, but the magnitude of the facility increase and morphologic changes were much less than in the living monkey. This supports the idea that inner wall separation from the JCT may modulate outflow facility.

Actin structure in the outflow tract of normal and glaucomatous eyes.

PURPOSE: To characterize the in situ distribution of actin in Schlemm's canal endothelium (SCE) and juxtacanalicular tissue (JCT) cells in glaucomatous human eyes, and compare to the distribution in normal eyes. METHODS: Fresh human eye bank eyes were perfused and fixed at pressure (n=27 normal eyes and 22 confirmed glaucomatous eyes). Schlemm's canal was opened by microdissection and outflow tissues were labelled for confocal microscopy to visualize F-actin, nuclei, laminin and/or CD31. Images were acquired in Z-series from the inner wall of Schlemm's canal, juxtacanalicular tissue and outer corneoscleral meshwork. RESULTS: In normal eyes, inner wall Schlemm's canal endothelial (SCE) cells showed a dense peripheral F-actin band, as previously described. JCT cells showed a more random and amorphous F-actin distribution. In glaucoma eyes, peripheral F-actin bands were less common in inner wall SCE cells; instead, F-actin was more centrally located within the cell and appeared 'tangled'. These actin tangles were also prominent in JCT cells of glaucoma eyes. Glaucoma eyes also demonstrated structures with features of cross-linked actin networks (CLANs), and more frequent occurrence of punctuate actin concentrations. There was a significant degree of heterogeneity, with some regions from glaucomatous eyes appearing normal and vice versa. CONCLUSION: F-actin architecture in human outflow pathway cells in situ differs between normal and glaucoma eyes, with glaucomatous tissue showing a more 'disordered' actin architecture overall. Some of these changes are likely due to effects secondary to administration of anti-glaucoma medications. Most of the changes that we observed could potentially affect the biomechanical properties of the outflow pathway tissues in glaucoma, but their role in the pathogenesis of ocular hypertension remains unclear.

Perfusion with the olfactomedin domain of myocilin does not affect outflow facility.

PURPOSE: Mutations in the MYOC gene coding for myocilin are associated with elevated intraocular pressure (IOP), and recombinant myocilin, produced in a prokaryotic expression system, has been reported to affect aqueous outflow facility. This study was conducted to test whether perfusion with a fragment of recombinant myocilin (containing the full-length olfactomedin domain), produced in a eukaryotic expression system, affects facility. METHODS: 293 EBNA cells were transfected by a vector containing the BM40 signal peptide, a human cDNA coding for myocilin, and a polyhistidine tag (HisTag) sequence. Recombinant protein was isolated by Ni-chelate chromatography, and characterized, and perfused into cultured anterior segments of human and porcine eyes. RESULTS: Recombinant myocilin was secreted as a approximately 55-kDa intact protein and two fragments arising from cleavage of the recombinant protein at amino acid 215. The C-terminal fragment, containing the entire olfactomedin domain, was successfully isolated. When perfused into human and porcine eyes, this C-terminal fragment did not appreciably affect outflow facility. CONCLUSIONS: Although the olfactomedin domain appears to be important for the function of myocilin, perfusion with a recombinant myocilin fragment containing this domain does not change outflow facility. It is possible that both the olfactomedin and N-terminal domains (including the leucine zipper) must be present for myocilin to have full function. Alternatively, posttranslational modifications of myocilin may have a major impact on protein function.