Trabecular Meshwork Stiffness Research Articles

The Factors Affecting the Stability of IOP Homeostasis.

Shear-induced nitric oxide (NO) production by Schlemm's canal (SC) endothelial cells provides a fast, IOP-sensitive feedback signal that normally contributes to IOP homeostasis. Our goal was to analyze the response of this homeostatic system under constant flow perfusion (as occurs in vivo) vs. constant pressure perfusion (as typical for laboratory perfusions). A mathematical model of aqueous humor dynamics, including shear-mediated NO signaling, was formulated and analyzed for stability. The model includes Goldmann's equation, accounting for proximal and distal outflow resistance, and describes how elevated IOP causes narrowing of SC lumen that increases the shear stress on SC cells. Elevated shear stress stimulates NO production, which acts to reduce outflow resistance and relax trabecular meshwork cells to decrease trabecular meshwork stiffness, affecting the SC luminal caliber. During constant flow perfusion, the outflow system is typically stable, returning to baseline IOP after a perturbation. In contrast, during constant pressure perfusion, the outflow system can become unstable and exhibit a time-dependent change in outflow resistance that diverges from baseline. The stability of shear mediated IOP homeostasis is predicted to differ critically between constant flow vs. constant pressure perfusion. Because outflow facility is typically measured at a constant pressure in the laboratory, this instability may contribute to the characteristic time-dependent increase in outflow facility, known as washout, observed in many nonhuman species. Studies of IOP homeostasis should consider how the outflow system may respond differently under constant pressure vs. constant flow perfusion.

Sigma-1 Receptor Activation Is Protective against TGFβ2-Induced Extracellular Matrix Changes in Human Trabecular Meshwork Cells.

The trabecular meshwork (TM) route is the principal outflow egress of the aqueous humor. Actin cytoskeletal remodeling in the TM and extracellular matrix (ECM) deposition increase TM stiffness, outflow resistance, and elevate intraocular pressure (IOP). These alterations are strongly linked to transforming growth factor-β2 (TGFβ2), a known profibrotic cytokine that is markedly elevated in the aqueous humor of glaucomatous eyes. Sigma-1 receptor (S1R) has been shown to have neuroprotective effects in the retina, but data are lacking about its role in the TM. In this study, we identified the presence of S1R in mouse TM tissue and investigated the effect of an S1R agonist fluvoxamine (FLU) on TGFβ2-induced human TM cells regarding cell proliferation; ECM-related functions, including F-actin reorganization; and the accumulation of ECM elements. TGFβ2 increased the proliferation, cytoskeletal remodeling, and protein levels of fibronectin, collagen type IV, and connective tissue growth factor, and decreased the level of matrix metalloproteinase-2. Most importantly, FLU reversed all these effects of TGFβ2, suggesting that S1R agonists could be potential candidates for preserving TM function and thus maintaining normal IOP.

Multiomics analysis reveals the mechanical stress-dependent changes in trabecular meshwork cytoskeletal-extracellular matrix interactions.

Trabecular meshwork (TM) tissue is subjected to constant mechanical stress due to the ocular pulse created by the cardiac cycle. This brings about alterations in the membrane lipids and associated cell–cell adhesion and cell–extracellular matrix (ECM) interactions, triggering intracellular signaling responses to counter mechanical insults. A loss of such response can lead to elevated intraocular pressure (IOP), a major risk factor for primary open-angle glaucoma. This study is aimed to understand the changes in signaling responses by TM subjected to mechanical stretch. We utilized multiomics to perform an unbiased mRNA sequencing to identify changes in transcripts, mass spectrometry- (MS-) based quantitative proteomics for protein changes, and multiple reaction monitoring (MRM) profiling-based MS and high-performance liquid chromatography (HPLC-) based MS to characterize the lipid changes. We performed pathway analysis to obtain an integrated map of TM response to mechanical stretch. The human TM cells subjected to mechanical stretch demonstrated an upregulation of protein quality control, oxidative damage response, pro-autophagic signal, induction of anti-apoptotic, and survival signaling. We propose that mechanical stretch-induced lipid signaling via increased ceramide and sphingomyelin potentially contributes to increased TM stiffness through actin-cytoskeleton reorganization and profibrotic response. Interestingly, increased phospholipids and diacylglycerol due to mechanical stretch potentially enable cell membrane remodeling and changes in signaling pathways to alter cellular contractility. Overall, we propose the mechanistic interplay of macromolecules to bring about a concerted cellular response in TM cells to achieve mechanotransduction and IOP regulation when TM cells undergo mechanical stretch.

Biomechanics of human trabecular meshwork in healthy and glaucoma eyes via dynamic Schlemm's canal pressurization

Background and objectiveThe trabecular meshwork (TM) consists of extracellular matrix (ECM) with embedded collagen and elastin fibers providing its mechanical support. TM stiffness is considerably higher in glaucoma eyes. Emerging data indicates that the TM moves dynamically with transient intraocular pressure (IOP) fluctuations, implying the viscoelastic mechanical behavior of the TM. However, little is known about TM viscoelastic behavior. We calculated the viscoelastic mechanical properties of the TM in n = 2 healthy and n = 2 glaucoma eyes. MethodsA quadrant of the anterior segment was submerged in a saline bath, and a cannula connected to an adjustable saline reservoir was inserted into Schlemm's canal (SC). A spectral domain-OCT (SD-OCT) provided continuous cross-sectional B-scans of the TM/JCT/SC complex during pressure oscillation from 0 to 30 mmHg at two locations. The TM/JCT/SC complex boundaries were delineated to construct a 20-µm-thick volume finite element (FE) mesh. Pre-tensioned collagen and elastin fibrils were embedded in the model using a mesh-free penalty-based cable-in-solid algorithm. SC pressure was represented by a position- and time-dependent pressure boundary; floating boundary conditions were applied to the other cut edges of the model. An FE-optimization algorithm was used to adjust the ECM/fiber mechanical properties such that the TM/JCT/SC model and SD-OCT imaging data best matched over time. ResultsSignificantly larger short- and long-time ECM shear moduli (p = 0.0032), and collagen (1.82x) and elastin (2.72x) fibril elastic moduli (p = 0.0001), were found in the TM of glaucoma eyes compared to healthy controls. ConclusionsThese findings provide additional clarity on the mechanical property differences in healthy and glaucomatous outflow pathway under dynamic loading. Understanding the viscoelastic properties of the TM may serve as a new biomarker in early diagnosis of glaucoma.

Cross-linked actin networks (CLANs) affect stiffness and/or actin dynamics in transgenic transformed and primary human trabecular meshwork cells

Cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells may contribute to increased IOP by altering TM cell function and stiffness. However, there is a lack of direct evidence. Here, we developed transformed TM cells that form spontaneous fluorescently labelled CLANs. The stable cells were constructed by transducing transformed glaucomatous TM (GTM3) cells with the pLenti-LifeAct-EGFP-BlastR lentiviral vector and selection with blasticidin. The stiffness of the GTM3-LifeAct-GFP cells were studied using atomic force microscopy. Elastic moduli of CLANs in primary human TM cells treated with/without dexamethasone/TGFβ2 were also measured to validate findings in GTM3-LifeAct-GFP cells. Live-cell imaging was performed on GTM3-LifeAct-GFP cells treated with 1 μM latrunculin B or pHrodo bioparticles to determine actin stability and phagocytosis, respectively. The GTM3-LifeAct-GFP cells formed spontaneous CLANs without the induction of TGFβ2 or dexamethasone. The CLAN containing cells showed elevated cell stiffness, resistance to latrunculin B-induced actin depolymerization, as well as compromised phagocytosis, compared to the cells without CLANs. Primary human TM cells with dexamethasone or TGFβ2-induced CLANs were also stiffer and less phagocytic. The GTM3-LifeAct-GFP cells are a novel tool for studying the mechanobiology and pathology of CLANs in the TM. Initial characterization of these cells showed that CLANs contribute to at least some glaucomatous phenotypes of TM cells.

Pulsatile Trabecular Meshwork Motion: An Indicator of Intraocular Pressure Control in Primary Open-Angle Glaucoma.

(1) Background: To investigate the value of pulsatile trabecular meshwork (TM) motion in predicting the diurnal intraocular pressure (IOP) fluctuation of primary open-angle glaucoma (POAG). (2) Methods: This cross-sectional study recruited 20 normal patients and 30 patients with POAG. Of the POAG group, 20 had stable diurnal IOP and 10 had high IOP fluctuation. A clinical prototype phase-sensitive optical coherence tomography (PhS-OCT) model was used to measure TM pulsatile motion with maximum velocity (MV) and cumulative displacement (CDisp). (3) Results: MV and CDisp were higher in the external region in both normal and POAG patients. All MV and CDisp reduced significantly in the POAG group (p < 0.001). In the POAG group, except MV in the external region (p = 0.085), MV and CDisp in the nasal area were significantly higher than those in the temporal area (p < 0.05). The MV and CDisp in the external region in the nasal area of POAG patients with high IOP fluctuation were much lower than those with stable IOP (pEMV3 = 0.031, pECDisp3 < 0.001); (4) Conclusions: Pulsatile TM motion reduced in POAG patients relevant to the level of diurnal IOP fluctuation. This study presents the segmental variance of TM stiffness in human living eyes and suggests the clinical potential of the measurement of pulsatile TM motion with PhS-OCT for the evaluation of diurnal IOP fluctuation.

Endogenous expression of Notch pathway molecules in human trabecular meshwork cells

PurposeCells in the trabecular meshwork sense and respond to a myriad of physical forces through a process known as mechanotransduction. Whilst the effect of substratum stiffness or stretch on TM cells have been investigated in the context of transforming growth factor (TGF-β), Wnt and YAP/TAZ pathways, the role of Notch signaling, an evolutionarily conserved pathway, recently implicated in mechanotransduction, has not been investigated in trabecular meshwork (TM) cells. Here, we compare the endogenous expression of Notch pathway molecules in TM cells from glaucomatous and non-glaucomatous donors, segmental flow regions, and when subjected to cyclical strain, or grown on hydrogels of varying rigidity. MethodsPrimary TM from glaucomatous (GTM), non-glaucomatous (NTM) donors, and from segmental flow regions [high flow (HF), low flow (LF)], were utilized between passages 2–6. Cells were (i) plated on tissue culture plastic, (ii) subjected to cyclical strain (6 h and 24 h), or (iii) cultured on 3 kPa and 80 kPa hydrogels. mRNA levels of Notch receptors/ligands/effectors in the TM cells was determined by qRT-PCR. Phagocytosis was determined as a function of substratum stiffness in NTM-HF/LF cells in the presence or absence of 100 nM Dexamethasone treatment. ResultsInnate expression of Notch pathway genes were significantly overexpressed in GTM cells with no discernible differences observed between HF/LF cells in either NTM or GTM cells cultured on plastic substrates. With 6 h of cyclical strain, a subset of Notch pathway genes presented with altered expression. Expression of Notch receptors/ligands/receptors/inhibitors progressively declined with increasing stiffness and this correlated with phagocytic ability of NTM cells. Dexamethasone treatment decreased phagocytosis regardless of stiffness or cells isolated from segmental outflow regions. ConclusionsWe demonstrate here that the Notch expression in cultured TM cells differ intrinsically between GTM vs NTM, and by substratum cues (cyclical strain and stiffness). Of import, the most apparent differences in gene expression were observed as a function of substratum stiffness which closely followed phagocytic ability of cells. Interestingly, on soft substrates (mimicking normal TM stiffness) Notch expression and phagocytosis was highest, while both expression and phagocytosis was significantly lower on stiffer substrates (mimicking glaucomatous stiffness) regardless of DEX treatment. Such context dependent changes suggest Notch pathway may play differing roles in disease vs homeostasis. Studies focused on understanding the mechanistic role of Notch (if any) in outflow homeostasis are thus warranted.

Determination of the Role of the Distal Outflow Pathway Tissue in Glucocorticoid-induced Ocular Hypertension

Introduction&#x0D; Prolonged application of glucocorticoids (GCs) induces ocular hypertension (OHT) and glaucoma. This increased intraocular pressure (IOP) is due to pathological changes in the trabecular meshwork (TM) outflow pathway tissues including impaired cell functions and extracellular matrix deposition. The changes and role of the TM in GC-induced OHT have been well studied. However, the role of the tissues distal to the TM (distal outflow tissues) is unclear. This study aims to further uncover the role of distal outflow tissue in GC-induced OHT using a novel perfusion organ culture (POC) model.&#x0D; Methods&#x0D; Huma corneal rims tissues were attached to 3D printed transparent perfusion plates using a combination of thin and thick glues. The artificial anterior chamber was perfused with DMEM-low glucose medium at 2ul/min to mimic aqueous humor production, and IOP was recorded using pressure transducers and a computerized system. To determine the role of distal tissue in GC-induced IOP changes, the TM tissue was carefully removed from both eyes, and one eye was treated with ethanol (EtOH) and the fellow eye with dexamethasone (DEX).&#x0D; Results&#x0D; The model was validated through a comparison of the IOP and TM stiffness of glue contaminated to non-contaminated corneal rims. The glue contaminated rim showed highly increased IOP and TM stiffness while the non-contaminated rim showed normal values. After validation, the TM was removed from paired corneal rims. One rim was treated with 100nM DEX and the fellow rim with 0.1% EtOH. The DEX treated rim showed increase in IOP while the EtOH control showed little change.&#x0D; Conclusion&#x0D; We created a novel corneal rim perfusion culture model for the study of GC-induced OHT. This model showed promising results of distal outflow involvement in glucocorticoid induced ocular hypertension. Further studies are needed to elucidate the role of distal outflow tissues in GC responsiveness in the eye.

An ex vivo model of human corneal rim perfusion organ culture

The human anterior segment perfusion culture model is a valuable tool for studying the trabecular meshwork (TM) and aqueous humor outflow in glaucoma. The traditional model relies on whole eye globes resulting in high cost and limited availability. Here, we developed a glue-based method which enabled us to use human corneal rims for perfusion culture experiments. Human corneal rim perfusion culture plates were 3D printed. Human corneal rims containing intact TM were attached and sealed to the plate using low viscosity and high viscosity glues, respectively. The human corneal rims were perfused using the constant flow mode, and the pressure changes were recorded using a computerized system. Outflow facility, TM stiffness, and TM morphology were evaluated. When perfused at rates from 1.2 to 3.6 μl/min, the outflow facility was 0.359 ± 0.216 μl/min/mmHg among 10 human corneal rims. The stiffness of the TM in naïve human corneal rim was similar to that of perfusion cultured human corneal rim. Also, the stiffness of TM of corneal rims perfused with dexamethasone was significantly higher than the control. Human corneal rims with glue contamination in the TM could be differentiated by high baseline intraocular pressure as well as high TM stiffness. Histology studies showed that the TM tissues perfused with plain medium appeared normal. We believed that our glued-based method is a useful tool and low-cost alternative to the traditional anterior segment perfusion culture model.

Replacement of the Trabecular Meshwork Cells-A Way Ahead in IOP Control?

Glaucoma is one of the leading causes of vision loss worldwide, characterised with irreversible optic nerve damage and progressive vision loss. Primary open-angle glaucoma (POAG) is a subset of glaucoma, characterised by normal anterior chamber angle and raised intraocular pressure (IOP). Reducing IOP is the main modifiable factor in the treatment of POAG, and the trabecular meshwork (TM) is the primary site of aqueous humour outflow (AH) and the resistance to outflow. The structure and the composition of the TM are key to its function in regulating AH outflow. Dysfunction and loss of the TM cells found in the natural ageing process and more so in POAG can cause abnormal extracellular matrix (ECM) accumulation, increased TM stiffness, and increased IOP. Therefore, repair or regeneration of TM’s structure and function is considered as a potential treatment for POAG. Cell transplantation is an attractive option to repopulate the TM cells in POAG, but to develop a cell replacement approach, various challenges are still to be addressed. The choice of cell replacement covers autologous or allogenic approaches, which led to investigations into TM progenitor cells, induced pluripotent stem cells (iPSCs), and mesenchymal stem cells (MSCs) as potential stem cell source candidates. However, the potential plasticity and the lack of definitive cell markers for the progenitor and the TM cell population compound the biological challenge. Morphological and differential gene expression of TM cells located within different regions of the TM may give rise to different cell replacement or regenerative approaches. As such, this review describes the different approaches taken to date investigating different cell sources and their differing cell isolation and differentiation methodologies. In addition, we highlighted how these approaches were evaluated in different animal and ex vivo model systems and the potential of these methods in future POAG treatment.

Mechanotransduction in trabecular meshwork cells requires obligatory interactions between TRPV4 channels, Rho signaling and focal adhesions

PurposeSustained exposure to mechanical forces triggers cellular remodeling that is essential to protect cells against mechanical stress. While this process is adaptive in the short term, chronic increases in cellular stiffness are paradigmatic of heart, lung, brain and eye diseases, however, the molecular mechanisms are not well understood. Here, we investigate the reciprocal interactions between cellular mechanotransducers (mechanosensitive ion channels and integrin‐based focal adhesions), calcium signaling, Rho signaling and actin remodeling in trabecular meshwork (TM) cells, which control intraocular pressure (IOP) via a feedback loop between IOP‐induced tensile stretch, cell stiffness and contractility. This process drives vision loss in glaucoma but the mechanisms are unknown.MethodsJuxtacanalicular and corneoscleral human TM cells were stimulated with biaxial strain (6–12%) for 1–24 hours in the presence of agonists/blockers of mechanosensitive channels, calcium chelators and/or Rho kinase inhibitors. [Ca2+] levels in TM cells were measured using optical imaging or electrophysiology. Live imaging cells were transfected mApple:actin or mCherry:actin. mRNA and protein levels of cytoskeletal, ECM and focal adhesion molecules were determined with qRT‐PCR, Western blot and immunostaining.ResultsExposure to strain and targeted activation of TRPV4 resulted in dramatic increase in F‐actin polymerization, which required calcium influx via TRPV4 channels. Stretch time‐dependently upregulated the transcript and protein levels of multiple focal adhesion (FA) proteins that included paxillin, focal adhesion kinase (FAK) and zyxin, an effect that was associated with phosphorylation of FA adapter/anchoring proteins and their translocation between cytoskeletal and membrane locations. Stretch‐induced, Rho‐dependent actin polymerization was downstream from TRPV4 channel activation and visualized as increased formation of stress fibers, however, not all of the intermediary steps were Ca2+‐dependent. Moreover, mechanical stress increased the size of FAs, facilitated vinculin colocalization with pFAK, and triggered zyxin relocation in a Ca2+‐dependent manner. Suppression of mechanotransduction mechanisms reduced actin polymerization, decreased cell contractility and increased fluid outflow.Summary/conclusionWe identified TRPV4 channels as key drivers of strain‐dependent cellular remodeling in the TM, and showed that this process plays a fundamental role in IOP regulation. TRPV4‐mediated Ca2+ influx was required to drive mechanoreciprocity – force‐dependent remodeling of FA and actin – to adapt the cells to mechanical stress. This time‐dependent mechanism appears to be required for the dynamic sensing of membrane strain and its role in adjusting steady‐state IOP. In glaucoma, this process is derailed by chronic increases in actin polymerization and TM stiffness that compromise fluid outflow from the anterior eye.Support or Funding InformationAcknowledgmentsFunding sourceThis work was supported by NIH (RO1EY027920, RO1EY022076, P30EY014800), Glaucoma Research Foundation and unrestricted grants from Research to Prevent Blindness to the Moran Eye Institute at the University of Utah.

In vivo measurement of trabecular meshwork stiffness in a corticosteroid-induced ocular hypertensive mouse model

Ocular corticosteroids are commonly used clinically. Unfortunately, their administration frequently leads to ocular hypertension, i.e., elevated intraocular pressure (IOP), which, in turn, can progress to a form of glaucoma known as steroid-induced glaucoma. The pathophysiology of this condition is poorly understood yet shares similarities with the most common form of glaucoma. Using nanotechnology, we created a mouse model of corticosteroid-induced ocular hypertension. This model functionally and morphologically resembles human ocular hypertension, having titratable, robust, and sustained IOPs caused by increased resistance to aqueous humor outflow. Using this model, we then interrogated the biomechanical properties of the trabecular meshwork (TM), including the inner wall of Schlemm's canal (SC), tissues known to strongly influence IOP and to be altered in other forms of glaucoma. Specifically, using spectral domain optical coherence tomography, we observed that SC in corticosteroid-treated mice was more resistant to collapse at elevated IOPs, reflecting increased TM stiffness determined by inverse finite element modeling. Our noninvasive approach to monitoring TM stiffness in vivo is applicable to other forms of glaucoma and has significant potential to monitor TM function and thus positively affect the clinical care of glaucoma, the leading cause of irreversible blindness worldwide.

A model of the oscillatory mechanical forces in the conventional outflow pathway.

Intraocular pressure is regulated by mechanosensitive cells within the conventional outflow pathway, the primary route of aqueous humour drainage from the eye. However, the characteristics of the forces acting on those cells are poorly understood. We develop a model that describes flow through the conventional outflow pathway, including the trabecular meshwork (TM) and Schlemm’s canal (SC). Accounting for the ocular pulse, we estimate the time-varying shear stress on SC endothelium and strain on the TM. We consider a range of outflow resistances spanning normotensive to hypertensive conditions. Over this range, the SC shear stress increases significantly and becomes highly oscillatory. TM strain also increases, but with negligible oscillations. Interestingly, TM strain responds more to changes in outflow resistance around physiological values, while SC shear stress responds more to elevated levels of resistance. A modest increase in TM stiffness, as observed in glaucoma, suppresses TM strain and practically eliminates the influence of outflow resistance on SC shear stress. As SC and TM cells respond to mechanical stimulation by secreting factors that modulate outflow resistance, our model provides insight regarding the potential role of SC shear and TM strain as mechanosensory cues for homeostatic regulation of outflow resistance and hence intraocular pressure.

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.

Estimating Human Trabecular Meshwork Stiffness by Numerical Modeling and Advanced OCT Imaging.

PurposeThe purpose of this study was to estimate human trabecular meshwork (hTM) stiffness, thought to be elevated in glaucoma, using a novel indirect approach, and to compare results with direct en face atomic force microscopy (AFM) measurements.MethodsPostmortem human eyes were perfused to measure outflow facility and identify high- and low-flow regions (HF, LF) by tracer. Optical coherence tomography (OCT) images were obtained as Schlemm's canal luminal pressure was directly manipulated. TM stiffness was deduced by an inverse finite element modeling (FEM) approach. A series of AFM forcemaps was acquired along a line traversing the anterior angle on a radially cut flat-mount corneoscleral wedge with TM facing upward.ResultsThe elastic modulus of normal hTM estimated by inverse FEM was 70 ± 20 kPa (mean ± SD), whereas glaucomatous hTM was slightly stiffer (98 ± 19 kPa). This trend was consistent with TM stiffnesses measured by AFM: normal hTM stiffness = 1.37 ± 0.56 kPa, which was lower than glaucomatous hTM stiffness (2.75 ± 1.19 kPa). None of these differences were statistically significant. TM in HF wedges was softer than that in LF wedges for both normal and glaucomatous eyes based on the inverse FEM approach but not by AFM. Outflow facility was significantly correlated with TM stiffness estimated by FEM in six human eyes (P = 0.018).ConclusionsTM stiffness is higher, but only modestly so, in glaucomatous patients. Outflow facility in both normal and glaucomatous human eyes appears to associate with TM stiffness. This evidence motivates further studies to investigate factors underlying TM biomechanical property regulation.

Stiffness characterization of anisotropic trabecular meshwork

Elevation of intraocular pressure has been correlated to changes in stiffness of trabecular meshwork (TM) in glaucomatous eyes although mechanical properties of the TM remain to be quantitatively determined. Data in the literature suggest that the TM cannot be considered mechanically as a uniform layer of isotropic elastic material, because the value of its Young’s modulus depends on the methods of measurements and can vary up to five orders of magnitude. To this end, we proposed a new theoretical framework for mechanical analysis of the TM, in which the inner wall of Schlemm’s canal and the juxtacanalicular tissue in the TM were treated as a uniform layer of isotropic elastic material, and the rest of the TM, i.e., the uveal and corneoscleral meshworks, were modeled as a uniform layer of transversely isotropic material. Using the model, we demonstrated that the large discrepancy in the apparent Young’s modulus reported in the literature could be caused by the anisotropy of the meshwork that was significantly stiffer in the longitudinal direction than in the transverse direction. The theoretical framework could be used to integrate existing data of the stiffness, investigate anisotropic behaviors of the tissues, and develop new methods to measure mechanical properties of the TM.

An imaged-based inverse finite element method to determine in-vivo mechanical properties of the human trabecular meshwork

Aim/Purpose: Previous studies have shown that the trabecular meshwork (TM) is mechanically stiffer in glaucomatous eyes as compared to normal eyes. It is believed that elevated TM stiffness increases resistance to the aqueous humor outflow, producing increased intraocular pressure (IOP).It would be advantageous to measure TM mechanical properties in vivo, as these properties are believed to play an important role in the pathophysiology of glaucoma and could be useful for identifying potential risk factors. The purpose of this study was to develop a method to estimate in-vivo TM mechanical properties using clinically available exams and computer simulations.Design: Inverse finite element simulationMethods: A finite element model of the TM was constructed from optical coherence tomography (OCT) images of a healthy volunteer before and during IOP elevation. An axisymmetric model of the TM was then constructed. Images of the TM at a baseline IOP level of 11, and elevated level of 23 mmHg were treated as the undeformed and deformed configurations, respectively. An inverse modeling technique was subsequently used to estimate the TM shear modulus (G). An optimization technique was used to find the shear modulus that minimized the difference between Schlemm’s canal area in the in-vivo images and simulations.Results: Upon completion of inverse finite element modeling, the simulated area of the Schlemm’s canal changed from 8,889 μm2 to 2,088 μm2, similar to the experimentally measured areal change of the canal (from 8,889 μm2 to 2,100 μm2). The calculated value of shear modulus was found to be 1.93 kPa, (implying an approximate Young’s modulus of 5.75 kPa), which is consistent with previous ex-vivo measurements.Conclusion: The combined imaging and computational simulation technique provides a unique approach to calculate the mechanical properties of the TM in vivo without any surgical intervention. Quantification of such mechanical properties will help us examine the mechanistic role of TM biomechanics in the regulation of IOP in healthy and glaucomatous eyes.

Dexamethasone Stiffens Trabecular Meshwork, Trabecular Meshwork Cells, and Matrix.

Treatment with corticosteroids can result in ocular hypertension and may lead to the development of steroid-induced glaucoma. The extent to which biomechanical changes in trabecular meshwork (TM) cells and extracellular matrix (ECM) contribute toward this dysfunction is poorly understood. Primary human TM (HTM) cells were cultured for either 3 days or 4 weeks in the presence or absence of dexamethasone (DEX), and cell mechanics, matrix mechanics and proteomics were determined, respectively. Adult rabbits were treated topically with either 0.1% DEX or vehicle over 3 weeks, and mechanics of the TM were determined. Treatment with DEX for 3 days resulted in a 2-fold increase in HTM cell stiffness, and this correlated with activation of extracellular signal-related kinase 1/2 (ERK1/2) and overexpression of α-smooth muscle actin (αSMA). Further, the matrix deposited by HTM cells chronically treated with DEX is approximately 4-fold stiffer, more organized, and has elevated expression of matrix proteins commonly implicated in glaucoma (decorin, myocilin, fibrillin, secreted frizzle-related protein [SFRP1], matrix-gla). Also, DEX treatment resulted in a 3.5-fold increase in stiffness of the rabbit TM. This integrated approach clearly demonstrates that DEX treatment increases TM cell stiffness concurrent with elevated αSMA expression and activation of the mitogen-activated protein kinase (MAPK) pathway, stiffens the ECM in vitro along with upregulation of Wnt antagonists and fibrotic markers embedded in a more organized matrix, and increases the stiffness of TM tissues in vivo. These results demonstrate glucocorticoid treatment can initiate the biophysical alteration associated with increased resistance to aqueous humor outflow and the resultant increase in IOP.

Trabecular Meshwork Response to Pressure Elevation in the Living Human Eye

The mechanical characteristics of the trabecular meshwork (TM) are linked to outflow resistance and intraocular pressure (IOP) regulation. The rationale behind this technique is the direct observation of the mechanical response of the TM to acute IOP elevation. Prior to scanning, IOP is measured at baseline and during IOP elevation. The limbus is scanned by spectral-domain optical coherence tomography at baseline and during IOP elevation (ophthalmodynamometer (ODM) applied at 30 g force). Scans are processed to enhance visualization of the aqueous humor outflow pathway using ImageJ. Vascular landmarks are used to identify corresponding locations in baseline and IOP elevation scan volumes. Schlemm canal (SC) cross-sectional area (SC-CSA) and SC length from anterior to posterior along its long axis are measured manually at 10 locations within a 1 mm segment of SC. Mean inner to outer wall distance (short axis length) is calculated as the area of SC divided by its long axis length. To examine the contribution of adjacent tissues to the effect IOP elevations, measurements are repeated without and with smooth muscle relaxation with instillation of tropicamide. TM migration into SC is resisted by TM stiffness, but is enhanced by the support of its attachment to adjacent smooth muscle within the ciliary body. This technique is the first to measure the living human TM response to pressure elevation in situ under physiological conditions within the human eye.

Regulation of Plasticity and Fibrogenic Activity of Trabecular Meshwork Cells by Rho GTPase Signaling

Glaucoma, a prevalent blinding disease is commonly associated with increased intraocular pressure due to impaired aqueous humor (AH) drainage through the trabecular meshwork (TM). Although increased TM tissue contraction and stiffness in association with accumulation of extracellular matrix (ECM) are believed to be partly responsible for increased resistance to AH outflow, the extracellular cues and intracellular mechanisms regulating TM cell contraction and ECM production are not well defined. This study tested the hypothesis that sustained activation of Rho GTPase signaling induced by lysophosphatidic acid (LPA), TGF-β, and connective tissue growth factor (CTGF) influences TM cell plasticity and fibrogenic activity which may eventually impact resistance to AH outflow. Various experiments performed using human TM cells revealed that constitutively active RhoA (RhoAV14), TGF-β2, LPA, and CTGF significantly increase the levels and expression of Fibroblast Specific Protein-1 (FSP-1), α-smooth muscle actin (αSMA), collagen-1A1 and secretory total collagen, as determined by q-RT-PCR, immunofluorescence, immunoblot, flow cytometry and the Sircol assay. Significantly, these changes appear to be mediated by Serum Response Factor (SRF), myocardin-related transcription factor (MRTF-A), Slug, and Twist-1, which are transcriptional regulators known to control cell plasticity, myofibroblast generation/activation and fibrogenic activity. Additionally, the Rho kinase inhibitor-Y27632 and anti-fibrotic agent-pirfenidone were both found to suppress the TGF-β2-induced expression of αSMA, FSP-1, and collagen-1A1. Taken together, these observations demonstrate the significance of RhoA/Rho kinase signaling in regulation of TM cell plasticity, fibrogenic activity, and myofibroblast activation, events with potential implications for the pathobiology of elevated intraocular pressure in glaucoma patients.