Accuracy of in vivo microCT imaging in assessing the microstructural properties of the mouse tibia subchondral bone.

Osteoarthritis (OA) is one of the most common musculoskeletal diseases. OA is characterized by degeneration of the articular cartilage as well as the underlying subchondral bone. Post-traumatic osteoarthritis (PTOA) is a subset of OA caused by mechanical trauma. Mouse models, such as destabilization of the medial meniscus (DMM), are useful to study PTOA. Ex vivo micro-Computed Tomography (microCT) imaging is the predominant technique used to scan the mouse knee in OA studies. Nevertheless, in vivo microCT enables the longitudinal assessment of bone microstructure, reducing measurement variability and number of animals required. The effect of image resolution in measuring subchondral bone parameters was previously evaluated only for a limited number of parameters. The aim of this study was to evaluate the ability of in vivo microCT imaging in measuring the microstructural properties of the mouse tibia trabecular and cortical subchondral bone, with respect to ex vivo high resolution imaging, in a DMM model of PTOA. Sixteen male C57BL/6J mice received DMM surgery or sham operation at 14 weeks of age (N=8 per group). The right knee of each mouse was microCT scanned in vivo (10.4µm voxel size) and ex vivo (4.35µm voxel size) at the age of 26 weeks. Each image was aligned to a reference image using rigid registration. The subchondral cortical bone plate thickness was measured at the lateral and medial condyles. Standard morphometric parameters were measured in the subchondral trabecular bone. In vivo microCT imaging led to significant underestimation of bone volume fraction (-14%), bone surface density (-3%) and trabecular number (-16%), whereas trabecular thickness (+3%) and separation (+5%) were significantly overestimated. Nevertheless, most trabecular parameters measured in vivo were well correlated with ex vivo measurements (R2 = 0.69-0.81). Degree of anisotropy, structure model index and connectivity density were measured in vivo with lower accuracy. Excellent accuracy was found for cortical thickness measurements. In conclusion, this study identified what bone morphological parameters can be reliably measured by in vivo microCT imaging of the subchondral bone in the mouse tibia. It highlights that this approach can be used to study longitudinal effects of diseases and treatments on the subchondral cortical bone and on most subchondral trabecular bone parameters, but systematic over- or under-estimations should be considered when interpreting the results.

Relationships between in vivo dynamic knee joint loading, static alignment and tibial subchondral bone microarchitecture in end-stage knee osteoarthritis.

OBJECTIVE: To study, in end-stage knee osteoarthritis (OA) patients, relationships between indices of in vivo dynamic knee joint loads obtained pre-operatively using gait analysis, static knee alignment, and the subchondral trabecular bone (STB) microarchitecture of their excised tibial plateau quantified with 3D micro-CT. DESIGN: Twenty-five knee OA patients scheduled for total knee arthroplasty underwent pre-operative gait analysis. Mechanical axis deviation (MAD) was determined radiographically. Following surgery, excised tibial plateaus were micro-CT-scanned and STB microarchitecture analysed in four subregions (anteromedial, posteromedial, anterolateral, posterolateral). Regional differences in STB microarchitecture and relationships between joint loading and microarchitecture were examined. RESULTS: STB microarchitecture differed among subregions (P < 0.001), anteromedially exhibiting highest bone volume fraction (BV/TV) and lowest structure model index (SMI). Anteromedial BV/TV and SMI correlated strongest with the peak external rotation moment (ERM; r = -0.74, r = 0.67, P < 0.01), despite ERM being the lowest (by factor of 10) of the moments considered, with majority of ERM measures below accuracy thresholds; medial-to-lateral BV/TV ratios correlated with ERM, MAD, knee adduction moment (KAM) and internal rotation moment (|r|-range: 0.54-0.74). When controlling for walking speed, KAM and MAD, the ERM explained additional 11-30% of the variations in anteromedial BV/TV and medial-to-lateral BV/TV ratio (R2 = 0.59, R2 = 0.69, P < 0.01). CONCLUSIONS: This preliminary study suggests significant associations between tibial plateau STB microarchitecture and knee joint loading indices in end-stage knee OA patients. Particularly, anteromedial BV/TV correlates strongest with ERM, whereas medial-to-lateral BV/TV ratio correlates strongest with indicators of medial-to-lateral joint loading (MAD, KAM) and rotational moments. However, associations with ERM should be interpreted with caution.

Joint loading and proximal tibia subchondral trabecular bone microarchitecture differ with walking gait patterns in end-stage knee osteoarthritis.

OBJECTIVES: To (1) stratify patient subgroups according to their distinct walking gait patterns in end-stage knee osteoarthritis (OA); (2) compare measures of joint loading and proximal tibia subchondral trabecular bone (STB) microarchitecture among these gait subgroups. DESIGN: Twenty-five knee OA patients undergoing total knee arthroplasty (TKA) had pre-operative gait analysis. Following surgery, excised tibial plateaus were micro-CT-scanned and STB microarchitecture analysed in four tibial condylar regions of interest. Peak knee moments were input to k-means cluster analysis, to identify subgroups with homogeneous gait patterns. Joint loading and STB microarchitecture parameters were compared among gait subgroups (Kruskal-Wallis, Bonferroni-corrected Mann-Whitney U tests). RESULTS: Three gait subgroups were revealed: biphasics (n = 7), flexors (n = 9), counter-rotators (n = 9). Peak knee adduction moment (KAM) and KAM impulse were significantly higher (P < 0.05) in biphasics than in flexors and counter-rotators (KAM = -0.65, -0.40 and -0.21 Nm/kg, respectively), suggesting a higher medial-to-lateral tibiofemoral load ratio in biphasics. Interestingly, STB medial-to-lateral bone volume fraction (BV/TV) ratio was also significantly higher (more than double) in biphasics and flexors than in counter-rotators (2.24, 2.00 and 1.00, respectively), whereas in biphasics it was only 10% higher than in flexors and not significantly so. CONCLUSIONS: Within the confines of the limited sample size, data suggests that different mechanisms between the biphasic and flexor gait subroups may generate comparable loads upon the tibial plateau and corresponding bony responses, despite significantly lower KAM indices in flexors. Hence, in flexor gait OA patients, conservative treatments designed to reduce KAM, may not be appropriate. Understanding joint loading among walking gait patterns and relationships to bone microarchitecture may aid at identifying/improving management of persons at risk for developing knee OA.

Coiled-coil targeting of myocilin to intracellular membranes.

Mutations in myocilin (MYOC) associate with glaucoma and ocular hypertension. Unfortunately, the specific role of MYOC, a widely expressed protein of unknown function, in ocular hypertension is unknown. Since MYOC localizes both to intracellular membranes and to the cytosol, we tested the hypothesis that MYOC is a cytosolic protein that associates with cellular membranes via its coiled-coil domain. Using green fluorescent protein (GFP) chimeras in expression and metabolic labeling studies, we observed that MYOC's putative signal peptide failed to traffic GFP into the secretory machinery and out of transfected cells. Next, we tested which of MYOC's three folding domains were responsible for targeting. In cell fractionation and immunofluorescence microscopy studies, the coiled-coil, but not the helix-turn-helix or olfactomedin domains, was necessary and sufficient to target GFP chimeras to cell membranes. Interestingly, a vesicular phenotype required sequential addition of the helix-turn-helix and olfactomedin domains to the coiled-coil. Taken together, these data indicate that the coiled-coil domain, not the putative signal sequence, is responsible for the targeting of MYOC to the secretory machinery.

Effect of hydrostatic pressure gradients and Na2EDTA on permeability of human Schlemm's canal cell monolayers.

PURPOSE: Elevated intraocular pressure in those with glaucoma appears to be a function of increased resistance to movement of aqueous humor through the conventional outflow pathway. The majority of resistance in both normal and glaucomatous eyes is generated in the region between the juxtacanalicular trabecular meshwork and the inner wall of Schlemm's canal. To accommodate transient elevations in pressure, we hypothesize that conventional outflow increases rapidly due to changes in complexity of intercellular junctions between cells of the inner wall of Schlemm's canal. METHODS: To test this hypothesis we examined specifically the effects of hydrostatic pressure gradients and the calcium chelator, Na2EDTA, on permeability of cultured human Schlemm's canal cell monolayers in isolation. Human Schlemm's Canal cells were isolated, cultured and then seeded onto permeable supports and maintained in culture to allow intercellular junctions to mature. With a minimum net transendothelial electrical resistance of 10 Ohm cm2, cells were placed into an Ussing-type chamber and hydraulic conductivity was calculated from pressure and flow measurements that were continuously recorded. Simultaneously, transendothelial electrical resistance was measured manually at fixed intervals. In parallel experiments, cell margins were monitored in real time by videomicroscopy. RESULTS: During the baseline measurement period when cells were exposed to pressure but not Na2EDTA, hydraulic conductivity was constant but transendothelial electrical resistance decreased continuously at rate of 0.24 Ohm cm2/minute. After Na2EDTA treatment, no significant change in transendothelial electrical resistance was measured while, hydraulic conductivity of Schlemm's Canal monolayers increased significantly by 125%; corresponding to noticeable intercellular separations. Restoration of cell-cell contact was observed by videomicroscopy 30 minutes following washout of Na2EDTA and functionally after 2 hours. CONCLUSIONS: Responses of Schlemm's Canal cells to pressure and calcium chelators in vitro are consistent with a role for calcium sensitive junctions in outflow resistance in vivo.

Expression of aquaporin-1 in human trabecular meshwork cells: role in resting cell volume.

PURPOSE: Drainage of aqueous humor from the human eye appears dependent on intracellular volume of trabecular meshwork (TM) cells, the predominant cell type of the human outflow pathway. Thus, the modulation of water and solute flux across the plasma membrane of TM cells is predicted to be an important factor in regulating outflow facility. Aquaporin (AQP)-1 is a hexahelical integral membrane protein that functions as a regulated channel for water and cations in fluid-secreting and -absorbing tissues. AQP1 is present in many tissues of the human eye, including the TM; however, its role in outflow facility is unknown. The purpose of the present study was twofold: to evaluate the prospect of manipulating AQP1 protein levels in TM cells using sense and antisense mRNA and to investigate the functional role of AQP1 in TM cells. METHODS: An adenovirus (AV) expression system was used to alter AQP1 protein levels. AQP1 protein expression was monitored using immunoblot analysis, and resting cell volume was measured by forward light scatter, electronic cell sizing, and [(14)C]-sucrose/urea equilibration. Permeability of TM monolayers to [(14)C]-sucrose was also assessed as an indirect evaluation of cell volume. RESULTS: AV-mediated gene transfer of AQP1 cDNA to TM cells resulted in a titer-dependent increase in recombinant AQP1, whereas transfer of antisense cDNA decreased native AQP1 protein by 71.7% +/- 5.5% (P < 0.01) after 5 days. A novel finding of this study is that mean resting volumes of AQP1(s) AV-infected TM cells in suspension were 8.7% +/- 3.0% greater (P < 0.05) than control cells. Conversely, AQP1 antisense (as) AV-infected cells had resting volumes 7.8% +/- 2.9% less than control cells (P < 0.05). Similar effects of AQP1 expression on resting cell volume were observed in TM monolayers. Consistent with this finding, paracellular permeability of AQP1(s) AV-infected TM monolayers to [(14)C]-sucrose decreased by 8.0% +/- 1.4% (P < 0.001). CONCLUSIONS: In addition to influencing the osmotic permeability of TM plasma membranes, the level of AQP1 protein expression influences resting intracellular volume and thus paracellular permeability of TM cell monolayers in vitro. These data suggest that AQP1 expression may affect outflow facility in vivo.

Isolation of primary open-angle glaucomatous trabecular meshwork cells from whole eye tissue.

PURPOSE: Isolation and culture of human trabecular meshwork (TM) cells from primary open-angle glaucomatous (POAG) tissue has proven difficult. The objective of this study was to directly compare the utility of two different isolation methods to obtain viable human TM cells from POAG whole eye tissue. METHODS: Using a blunt dissection technique, human TM tissue was obtained from four pairs of donor eyes (67, 77, 81 and 82 years) with a documented history of POAG. TM tissue from one eye was explanted into tissue culture. TM from the contralateral eye was digested with a collagenase mixture and seeded onto culture plates. RESULTS: Primary cell isolates were obtained from all donors with both techniques. However, only cells obtained using the digestion method (3 of 4 TMs) could be passaged for expansion and freeze-downs (3 x 107 second passage cells/donor). None of the cells obtained from explanted TMs could be passaged. Cells from successful isolations were of uniform size, possessed typical TM morphology and had doubling times < 48 hours. CONCLUSION: These results demonstrate a clear advantage to digesting the extracellular matrix of glaucomatous TM tissue to obtain sufficient numbers of healthy cells for use in experiments. In contrast to cells obtained from explants, cells liberated from POAG TM tissue by digestion appear indistinguishable morphologically and behaviorally from "normal" TM cells.

Hydraulic pressure stimulates adenosine 3',5'-cyclic monophosphate accumulation in endothelial cells from Schlemm's canal.

PURPOSE: Fluid flow across various endothelia results in a variety of intracellular and extracellular adaptations. In the living eye, aqueous humor flows across the surface of endothelial cells on trabecular meshwork (TM) beams and in the juxtacanalicular tissue and through or between a continuous monolayer of endothelial cells that line Schlemm's canal (SC). This study was undertaken to test the hypothesis that fluid flow induces biochemical changes in the endothelial cells of the outflow pathway that may modify outflow resistance. METHODS: Trabecular meshwork and SC cells isolated from the outflow pathway of human cadaveric eyes were seeded onto porous filters, placed in Ussing-type chambers, and subjected to fluid flow driven by a pressure head of 15 mm Hg on their apical surface. Cell lysates were prepared and analyzed for adenosine 3',5'-cyclic monophosphate (cAMP) accumulation. Barrier function of cell monolayers was examined using transendothelial electrical resistance measurements. RESULTS: Three different SC cell strains in 14 independent experiments responded with at least a threefold increase in cAMP that was both time and pressure dependent. Conversely, flow-treated TM cells failed to respond in six independent experiments in which five different TM cell strains were used. Electrical resistance across cell monolayers positively correlated with cAMP accumulation and was calcium sensitive. CONCLUSIONS: cAMP signaling is affected by pressure differentials across SC cell monolayers and provides evidence for the participation of SC cells in the regulation of aqueous outflow.

Acto-myosin drug effects and aqueous outflow function.

PURPOSE: Previous studies have identified the cytoskeletal proteins actin and tubulin as potential cellular targets in the trabecular meshwork for novel glaucoma therapy. The authors and others have hypothesized that acto-myosin interactions may be important for outflow function. The current study was conducted to evaluate 2,3-butanedione 2-monoxime (BDM), a compound that interferes with acto-myosin function through the myosin adenosine triphosphatase (ATPase) reaction; 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a proposed myosin light-chain kinase inhibitor; and the direct actin disrupter, latrunculin B, in an outflow pathway cell culture and perfused excised eye model system. METHODS: Freshly enucleated porcine eyes were perfused using the constant-pressure method at 15 mm Hg and 25 degrees C. Human trabecular meshwork (HTM) cells and Schlemm's canal (SC) cells were grown in culture, treated with BDM, H-7, and latrunculin B, and then fixed, stained for beta-tubulin and filamentous actin, and observed by epifluorescence. RESULTS: Twenty millimolar BDM, 100 microM H-7, and 1 microM latrunculin B increased outflow facility 36%, 63%, and 72%, respectively, compared to sham-treated controls, 13%, 15%, and 4% (n=7, 8, and 8; P=0.01, 0.0001, and 0.0002), respectively. In cultured HTM and SC cells, 100 microM H-7 caused a rapid loss of filamentous actin staining but did not produce a change in cell shape or cell- cell attachment. In contrast, 20 mM BDM induced a loss of cell- cell attachment and a change in cell shape that was associated with a 50% to 60% loss of filamentous actin staining, often in a distinct stick-and-ball pattern. Latrunculin B caused a severe loss of actin staining and cell shape changes. No drug altered beta-tubulin staining. CONCLUSIONS: Interference with myosin function can cause a secondary loss of actin organizational structure. Our study indicates that myosin, perhaps through its various phosphorylation reactions, may have a potential regulatory role in outflow function.

Isolation, culture, and characterization of endothelial cells from Schlemm's canal.

PURPOSE: An important goal in glaucoma research has been to understand the functional contribution of trabecular meshwork (TM) and Schlemm's canal (SC) endothelia to aqueous humor outflow resistance. To date, TM cells are routinely cultured and used as a model by several laboratories. However, there has been only limited success in isolating SC cells. The current objective was to develop a technique for selective isolation and culture of endothelial cells from human SC. METHODS: The anterior chamber of human cadaveric eyes was cut into eight equal and radially symmetric wedge-shaped pieces. Using a dissecting microscope, a gelatin-coated suture (6-0 sterile nylon monofilament) was gently inserted into the lumen of SC and advanced into the canal. The cannulated pieces of tissue were placed in culture medium and maintained for 3 weeks. Sutures were removed from SC and cells seeded onto 3-cm culture plates. Morphology, growth characteristics, and expression of endothelial surface antigens and other cellular markers were evaluated. RESULTS: Of the 20 pairs of eyes that were cannulated, primary cells were obtained from 13. All SC cell isolates had a fusiform morphology; formed nonoverlapping, linearly arranged monolayers; and were contact inhibited. Schlemm's canal cell isolates reacted with antibodies specific for CD44 (hyaluron receptor), CD54 (intercellular adhesion molecule-1, ICAM-1), tissue-type plasminogen activator, and TM-inducible glucocorticoid-responsive protein-myocilin (TIGR-MYOC). Unlike TM cells, however, TIGR-MYOC protein was not induced in SC cells after long-term dexamethasone treatment. Schlemm's canal cells endocytosed low-density lipoprotein and acetylated low-density lipoprotein, and in the presence of Matrigel organized into multicellular tubelike structures. CONCLUSIONS: Cannulation of SC with gelatin-coated suture material is an effective method for the isolation of human SC cells and provides a cellular model to study the potential role of SC cells in aqueous humor outflow function.

Microtubule disruption leads to cellular contraction in human trabecular meshwork cells.

PURPOSE: To determine whether microtubule- and actin-altering drugs, which have been shown to increase aqueous humor outflow, cause cellular contraction in human trabecular meshwork (HTM) cells. METHODS: HTM cells were plated in culture dishes containing a polymerized deformable silicone substrate. After 48 hours, the dishes were placed on an inverted microscope and treated with ethacrynic acid, colchicine, vinblastine, cytochalasin B, or 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and then recorded on videotape for 15 minutes. An increase in silicone substrate wrinkle size and/or number indicated a contraction. Sham controls were used. RESULTS: Cellular contraction was observed with ethacrynic acid, colchicine, and vinblastine in the 10(-5) to 10(-4) M dosage range. Pretreatment with H-7 blocked these effects. Cytochalasin B did not produce cellular contraction. CONCLUSIONS: Microtubule disruption causes cellular contraction in HTM cells, and this effect depends on an intact actin cytoskeleton network. Contraction of trabecular meshwork cells in response to various stimuli is an attractive hypothesis for possible homeostatic mechanisms in the outflow pathway, and this may serve as a focus for novel glaucoma drug development.

Nonsulfhydryl-reactive phenoxyacetic acids increase aqueous humor outflow facility.

PURPOSE: The phenoxyacetic acid, ethacrynic acid (ECA), has potential use in glaucoma therapy because it acts to increase aqueous outflow in vivo and in vitro. In human trabecular meshwork (HTM) cell culture, ECA acts to change cell shape and attachment, effects that have been correlated with microtubule (MT) alterations and chemical sulfhydryl (SH) reactivity. To further explore these actions, we evaluated two non-SH reactive phenoxyacetic acids, inadcrinone and ticrynafen, and the MT-disrupting drug vinblastine. METHODS: Excised bovine and porcine eyes were perfused and outflow facility measured. Calf pulmonary artery endothelial and HTM cells were grown in culture and cytoskeletal effects evaluated after drug treatment. RESULTS: Indacrinone, ticrynafen, and vinblastine all caused an increase in outflow facility. In contrast with ECA, the outflow effects of indacrinone and ticrynafen were not blocked by excess cysteine. Although indacrinone and ticrynafen produced changes in cell shape in vitro, the beta-tubulin staining pattern of treated cells was not altered. Vinblastine caused cell shape change and the expected MT disruption. CONCLUSIONS: Phenoxyacetic acids can increase aqueous outflow facility and alter HTM cell shape and attachment in vitro by a non-SH, non-MT mechanism (which is probably shared also by ECA). These findings suggest the possibility of a broader class of glaucoma drugs that may be directed at the HTM. An understanding of the cellular target for these drugs has implications both for potential glaucoma therapy and for the cytoskeletal mechanisms involved in normal outflow function.

Characterization of alpha 2-macroglobulin binding to human trabecular meshwork cells: presence of the alpha 2-macroglobulin signaling receptor.

Direct binding of receptor-recognized alpha 2-macroglobulin (alpha 2M*) or a cloned receptor binding fragment from rat alpha 1-macroglobulin (RBF) to human trabecular meshwork cells demonstrated two classes of cell surface binding sites. One class has an apparent Kd of 5.0 nM and a receptor number of 31,800 receptors/cell. The other class has an apparent Kd of 20 pM and a receptor number of 1600 receptors/cell. Binding studies of alpha 2M* or RBF in the presence of a competitor for binding to low-density-lipoprotein receptor-related protein/alpha 2M* receptor (LRP/alpha 2MR) called receptor-associated protein (RAP) show that only the lower affinity class of binding sites is susceptible to competition with RAP. Uptake studies demonstrate specific internalization and degradation of alpha 2M* which is inhibitable by RAP. Exposure of the cells to alpha 2M* and RBF (40 nM) is associated with mean increases of 171 and 210%, respectively, in the intracellular calcium concentration, which is not inhibitable by RAP or pertussis toxin. These studies present the first characterization of alpha 2M* and RBF signaling in a primary human cell type and suggest a role for alpha 2M* in the physiology of the eye.

Dexamethasone inhibits trabecular cell retraction.

Glucocorticosteroids such as dexamethasone (Dex) are known to cause an increased resistance to aqueous outflow in the intact and cultured eye. We investigated whether Dex treatment of cultured endothelial or trabecular meshwork (TM) cells might interfere with the cell separations and retraction induced by the facility-enhancing agents ethacrynic acid (ECA), cytochalasin B and the calcium chelator EGTA. Our hypothesis was that Dex-induced changes in the response of our model cells in vitro might serve as a paradigm for those produced in the cells of the outflow pathway, perhaps through influencing the changing dimensions of the pathway for aqueous humor through the juxtacanalicular tissue and/or inner wall of Schlemm's canal. We treated calf pulmonary artery endothelial (CPAE) and human and porcine TM cells with Dex (1-100 microns, 1-9 days), and then assessed monolayer and cytoskeletal integrity by immunofluorescence microscopy for tubulin and direct fluorescence staining for F-actin after exposure to the agents named above. We found that Dex-pretreated CPAE and TM cells gradually (over 5-7 days) became refractory to the effects of both ECA and EGTA, but not to cytochalasin B. Despite the preservation of general cell shape and attachment after ECA in Dex-treated cells, microtubule disruption still took place as in controls. Dex-treated cells also demonstrated a reorganization of filamentous actin staining after ECA and EGTA. Combination experiments of ECA and EGTA in Dex-treated cells suggested that the Dex effects were due to a greater strength of cell-to-cell and cell-to-substrate attachment, possibly due to interference with the normal cellular signaling required for coordinated cellular retraction and junctional disruption.

General surgical pre-admission clinic.

Ninety-nine patients from a non-urgent general surgical waiting list were randomly selected for either direct admission to a hospital bed or review at a preadmission clinic. A considerable reduction in subsequent bed occupancy was shown in the latter group. The findings suggest that more detailed review of patients in the outpatient department would result in the more efficient use of hospital facilities.