Purpose:Aqueous humor outflow through the trabecular meshwork (TM) is segmental, demonstrating high flow (HF) and low flow (LF) regions. Here, we investigate transcriptomic differences between flow regions in naïve mouse and non-glaucomatous human TM tissue to better understand intraocular pressure (IOP) regulation. Methods:Human eyes (<6 hours postmortem) from two donors were perfused with a fluorescent tracer to identify HF and LF regions and fixed. In parallel, one pair of 8-month-old C57Bl/6J mouse eyes were similarly processed. Sagittal sections from HF and LF regions underwent whole-transcriptome spatial profiling. Differential expression and gene set variation analysis were conducted to identify transcript and pathway-level differences between HF vs. LF TM. Selected targets were validated by immunolabeling. Results:Genes with relevance to TM outflow were identified as significantly differentially expressed in the human dataset, including ADAM metallopeptidase domain 15 (ADAM15), vimentin (VIM), chitinase 3-like 1 (CHI3L1), and several transcription factors (e.g., FOS, JUNB, ESR1). Pathways related to epigenetic modifications were also differentially enriched in human eyes. In both human and mouse eyes, myocilin was significantly upregulated in HF regions, despite greater protein labeling in LF regions of human eyes. In both species, rho-kinase signaling pathways showed increased enrichment in LF regions, while cell stress pathways, and TNF-α signaling were increased in HF TM. Conclusions:HF regions maintain a more active stress response that facilitates greater outflow, whereas LF regions exhibit more matrix accumulation and contractility. This characterization of segmental flow regions can inform future studies to target trabecular outflow and lower IOP.

Purpose: The lamina cribrosa (LC) is considered the initial site of glaucomatous retinal ganglion cell (RGC) injury, and is also the region where unmyelinated RGC axons become myelinated. Here we sought to use finite element (FE) modeling to investigate how the configuration of the myelination transition zone (MTZ) influences the mechanical insult to RGC axons. Methods: A multiscale FE framework was developed to investigate the biomechanical effect of myelin distribution on IOP-induced axonal stress and strain at the MTZ. An anatomically based macro-scale FE eye model was used to compute LC deformations under 15 and 45 mmHg IOP. These deformations were then applied to micro-scale models of the posterior LC, consisting of axons, myelin sheaths, and surrounding matrix. Four distinct MTZ boundary configurations were simulated: one flat and three with random posterior offsets of 3, 6, or 9 μm, representing potential physiological variations. IOP-induced effective axonal strains and stresses were quantified across the different MTZ configurations. Results: Under IOP loading, axons exhibited longitudinal compression and transverse stretch, with marked effective stress and strain discontinuities at the myelin boundary. Across all models, the unmyelinated region exhibited higher effective stress and strain than the myelinated region, and this mechanical discontinuity increased with larger MTZ offsets. Conclusions: Glaucoma-associated demyelination has been previously suggested to precede RNFL thinning. Here we have shown that the MTZ configuration directly influences RGC axonal mechanics. Whether different MTZ profiles can initiate glaucomatous injury, whether demyelination accelerates disease progression, or whether both mechanisms contribute, remains to be determined.

Purpose:Elevated IOP due to increased outflow resistance through the trabecular meshwork (TM) is a major risk factor for POAG. Outflow through the TM is segmental, consisting of high-flow (HF) and low-flow (LF) regions. Here, we investigate how ocular hypertension impacts segmental outflow using a dexamethasone (DEX) mouse model and compare TM stiffness between HF and LF regions. Methods:Nanoparticles containing DEX or vehicle were injected twice weekly in 2- to 4-month-old C57BL/6J mice (n = 14), and the IOP was measured weekly. At week 4, mouse eyes were perfused in vivo with fluorescent nanospheres to assess flow patterns and the circumferential percentage of HF, intermediate-flow, and LF regions in each eye. Sagittal sections were collected from HF and LF regions, and atomic force microscopy was used to measure tissue stiffness. Immunofluorescent labeling was used to compare fibronectin and alpha-smooth muscle actin protein levels. Results:DEX treatment significantly elevated the IOP by an average of 33.3% and altered tracer distribution but not the percentage of HF and LF regions around the circumference. No significant differences in TM stiffness were detected between DEX-treated and control mice, or between HF and LF regions. Increased fibronectin in LF regions of DEX-treated eyes suggested subtle TM structural changes that were not detected by atomic force microscopy. Conclusions:DEX alters segmental flow distribution and may impact cell contractility rather than ECM stiffness to cause IOP elevation in young mice. These findings better characterize the nature of segmental outflow and TM mechanics in this model of steroid-induced glaucoma.

Purpose:The inner wall of Schlemm's canal (SC) is a mechanosensitive endothelial monolayer that provides resistance to conventional aqueous humor drainage, a process dependent on pore formation. This study examined how microtubule (MT) stability affects SC cell mechanobiology, transcellular pore formation, and aqueous humor outflow dynamics. Methods:MT stability in cultured SC cells from normal and glaucomatous human donors was manipulated pharmacologically. Changes in MT acetylation, phosphorylated myosin light chain, and F-actin were assessed by immunofluorescence and immunoblotting. GEF-H1 was knocked down using siRNA. Cellular stiffness was measured by atomic force microscopy. Transcellular pore formation was quantified using an established pore formation assay. Outflow facility was measured in enucleated mouse eyes using the iPerfusion system. Results:MT stabilization in normal SC cells decreased actomyosin contractility and cellular stiffness, whereas MT destabilization increased contractility and stiffness; these effects involved the MT-associated Rho guanine nucleotide exchange factor GEF-H1. MT stability was also mechano-responsive to substrate stiffness. Furthermore, SC cells derived from glaucomatous donors exhibited reduced MT stability compared with normal SC cells. MT stabilization increased transcellular pore formation in both normal and glaucomatous SC cells. In ex vivo mouse eyes, paclitaxel perfusion to stabilize MTs significantly increased outflow facility relative to contralateral control eyes. Conclusions:Our data suggest that MT stability influences SC cell contractility, stiffness, and transcellular pore formation and can alter aqueous humor outflow facility. These findings identify MT-dependent cytoskeletal remodeling as an important contributor to the biomechanics of the conventional outflow pathway and suggest that MT-associated pathways may represent potential targets for improving outflow function in glaucoma.

PURPOSE. Cross-linked actin networks (CLANs) are prevalent in the glaucomatous trabecular meshwork (TM). We previously developed the GTM3L cell line, which spontaneously forms fluorescently labeled CLANs, by transducing GTM3, a transformed glaucomatous TM cell line, with a lentivirus expressing the LifeAct-GFP fusion protein. Here, we determined if LifeAct-GFP viral copy numbers are associated with CLANs, developed approaches to increase CLAN incidence, and computationally studied the biomechanical properties of CLAN-containing GTM3L cells. METHODS. GTM3L cells were fluorescently sorted for viral copy number analysis to determine whether increased CLAN incidence was associated with copy number. CLAN incidence was increased by combining (1) differential adhesion sorting, (2) cell deswelling, and (3) cell stiffness selection. GTM3L cells were cultured on glass or soft hydrogels for stiffness measurement by atomic force microscopy. Computational models studied the biomechanical properties of CLANs. RESULTS. GTM3L cells had one LifeAct-GFP viral copy/cell on average, and viral copy number or LifeAct-GFP expression level did not associate with CLAN incidence rate. However, CLAN rate was increased from -0.28% to -50% by combining the three enrichment methods noted above. Further, GTM3L cells formed more CLANs on a stiff versus a soft substrate. Computational modeling predicted that CLANs contribute to higher cell stiffness, including increased resistance of the nucleus to tensile stress when CLANs are physically linked to the nucleus. CONCLUSIONS. It is possible to greatly enhance CLAN incidence in GTM3L cells. CLANs are mechanosensitive structures that affect cell biomechanical properties. Further research is needed to determine the biomechanics, mechanobiology, and etiology of CLANs in the TM.

Purpose:Aqueous humor inflow rate, a key parameter influencing aqueous humor dynamics, is typically measured by fluorophotometry. Analyzing fluorophotometric data depends, inter alia, on the volume of aqueous humor in the anterior chamber but not the posterior chamber. Previous fluorophotometric studies of the aqueous inflow rate in mice have assumed the ratio of anterior:posterior volumes in mice to be similar to those in humans. Our goal was to measure anterior and posterior chamber volumes in mice to facilitate better estimates of aqueous inflow rates. Methods:We used standard near-infrared (NIR) optical coherence tomography (OCT) and robotic visible-light OCT (vis-OCT) to visualize, reconstruct, and quantify the volumes of the anterior and posterior chambers of the mouse eye in vivo. We used histology and micro-computed tomography (CT) scans to validate relevant landmarks from ex vivo tissues and facilitate in vivo measurement. Results:Posterior chamber volume is 1.1 times the anterior chamber volume in BALB/cAnNCrl mice, that is, the anterior chamber constitutes about 47% of the total aqueous humor volume, which is very dissimilar to the situation in humans. Anterior chamber volumes in 2-month-old BALB/cAnNCrl and C57BL6/J mice were 1.55 ± 0.36 µL (n = 10) and 2.05 ± 0.25 µL (n = 10), respectively. This implies that previous studies likely overestimated the aqueous inflow rate by approximately twofold. Conclusions:It is necessary to reassess previously reported estimates of aqueous inflow rates and, thus, aqueous humor dynamics in the mouse. For example, we now estimate that only 0% to 15% of aqueous humor drains via the pressure-independent (unconventional) route, similar to that seen in humans and monkeys.

Myopia, or near-sightedness, is rapidly growing in prevalence, with significant long-term implications for ocular health. There is thus great impetus to better understand molecular signalling pathways leading to myopia. We and others have reported that all-trans retinoic acid (atRA) is involved in myopigenic signalling, yet the understanding of how atRA is transported and exerts a myopigenic influence is poor. Here we measured the concentrations of atRA in the serum in wild-type C57BL/6 mice under control conditions and after atRA feeding, previously shown to induce myopia. We also developed a mathematical model that describes fluid fluxes and the advective-diffusive transport of atRA in choroid and sclera, including atRA synthesis in the choriocapillaris, atRA degradation by scleral cells, and binding of atRA to the carrier protein serum albumin. This model, developed for both mice and humans, showed that atRA produced in the choriocapillaris was able to permeate well into the sclera in both mice and humans at biologically relevant concentrations, and that atRA feeding greatly increased tissue levels of atRA across both the choroid and sclera. We were also able to identify which parameters most influence atRA concentration in ocular tissues, guiding future experimental work. Our findings support atRA's role in myopigenic signalling.

Purpose: Ambient light exposure is linked to myopia development in children and affects myopia susceptibility in animal models. Currently, it is unclear which signals mediate the effects of light on myopia. All-trans retinoic acid (atRA) and dopamine (DA) oppositely influence experimental myopia and may be involved in the retino-scleral signaling cascade underlying myopic eye growth. However, how ocular atRA responds to different lighting and whether atRA and DA interact remains unknown. Methods: Dark-adapted C57BL/6J mice (29-31 days old) were exposed to Dim (1 lux), Mid (59 lux), or Bright (12,000 lux) ambient lighting for 5-60 minutes. Some mice were also systemically administered the DA precursor, LDOPA, or atRA prior to light exposure. After exposure, the retina and the back-of-the-eye (BOE) were collected and analyzed for levels of atRA, DA, and the DA metabolite, DOPAC. Results: DA turnover (DOPAC/DA ratio) in the retina increased in magnitude after only five minutes of exposure to higher ambient luminance but was minimal in the BOE. In contrast, atRA levels in the retina and BOE significantly decreased with higher ambient luminance and longer duration exposure. Intriguingly, LDOPA-treated mice had a transient reduction in retinal atRA compared to saline-treated mice, whereas atRA treatment had no effect on ocular DA. Conclusions: Ocular atRA was affected by the duration of exposure to different ambient lighting and retinal atRA levels decreased with increased DA. Overall, these data suggest specific interactions between ambient lighting, atRA, and DA that could have implications for the retino-scleral signaling cascade underlying myopic eye growth.

Purpose: Myopia incidence is increasing globally. All-trans retinoic acid (atRA) is important in myopigenic retinoscleral signaling, motivating research on its ocular transport. However, atRA's weak autofluorescence limits its direct visualization in tissues. Further, atRA is hydrophobic and must bind to protein carriers for transport. We assessed a fluorescent analog of atRA (LightOx™14, CAS:198696-03-6, referred as 'floRA'), as an experimentally accessible atRA surrogate by: (i) evaluating its binding to carrier proteins and (ii) visualizing its distribution in ocular tissues. Methods: Binding: We assessed atRA-carrier protein binding using fluorescence quenching assays with bovine serum albumin (BSA), high density lipoprotein (HDL), apolipoprotein A-I (Apo A-I) and retinol binding protein 4 (RBP4). Direct visualization: Wild-type C57BL/6J mice were euthanized, eyes enucleated, and wedges containing sclera and choroid incubated for specific durations in 50 μM floRA+BSA. The wedge centers were cryo-sectioned and counterstained for nuclei. Fluorescent micrographs were acquired and analyzed using ImageJ. Results: Association constants (Ka) for atRA and floRA binding to carrier proteins were similar and ranged from 2-13 × 105 M-1, indicating non-specific binding. floRA could be visualized in sclera and choroid, yet showed significant spatial heterogeneity (enhanced fluorescence often colocalizing with nuclei). Conclusions: floRA is a reasonable surrogate for atRA binding to BSA, HDL, Apo A-I and RBP4. Considering these proteins' relative serum and extravascular abundances, and their similar binding affinity to atRA, we predict that serum albumin is an important atRA carrier. Use of floRA in whole tissue tracer studies shows promise but requires further refinement. ### Competing Interest Statement The authors have declared no competing interest.

We report to the readership of JBME the activities of JBME in 2024.

Trabecular meshwork (TM) cell therapy has been proposed as a next-generation treatment for elevated intraocular pressure (IOP) in glaucoma, the most common cause of irreversible blindness. Using a magnetic cell steering technique with excellent efficiency and tissue-specific targeting, we delivered two types of cells into a mouse model of glaucoma: either human adipose-derived mesenchymal stem cells (hAMSCs) or induced pluripotent cell derivatives (iPSC-TM cells). We observed a 4.5 [3.1, 6.0] mmHg or 27% reduction in intraocular pressure (IOP) for 9 months after a single dose of only 1500 magnetically steered hAMSCs, explained by increased outflow through the conventional pathway and associated with a higher TM cellularity. iPSC-TM cells were also effective, but less so, showing only a 1.9 [0.4, 3.3] mmHg or 13% IOP reduction and increased risk of tumorigenicity. In both cases, injected cells remained detectable in the iridocorneal angle 3 weeks post-transplantation. Based on the locations of the delivered cells, the mechanism of IOP lowering is most likely paracrine signaling. We conclude that magnetically steered hAMSC cell therapy has potential for long-term treatment of ocular hypertension in glaucoma.

The development of sophisticated computational tools, combined with advanced ultrastructural imaging techniques, offers unprecedented opportunities to investigate aqueous humor outflow through the conventional pathway, the site of pathology responsible for ocular hypertension in glaucoma. Recently, a series of studies1-7 have used computational methods to study conventional outflow function. Regrettably, these studies1-7 appear to contain fundamental errors that lead to predictions that are inconsistent with established outflow physiology and raise concerns regarding methodology and apparent misrepresentations of published work. As a result, this body of work draws potentially misleading and erroneous conclusions about aqueous humor dynamics. It is therefore important to recognize and discuss these works to correct the archival record and avoid misdirecting future research.

The eye presents a very dynamic biomechanical environment, and thus ocular cells must be highly mechanosensitive and mechanoresponsive. Moreover, defects in mechanobiological pathways contribute to a number of sight-threatening ocular diseases, highlighting the importance of ocular mechanobiology. We here give a concise overview of the mechanobiology of ocular cells in the lens and cornea (and how mechanobiology plays a role in associated pathologies in these tissues), before providing a detailed review of the mechanobiology of the common blinding disease, glaucoma. Mechanical stimuli are intimately linked with the pathology of glaucoma, both in terms of altered homeostasis of the eye's internal pressure control system and in the response of neural cells to elevated pressure in the eye. A complex array of mechanosensory elements (stretch-activated ion channels, integrins, G protein-coupled receptors) work together with intersecting networks of mechanotransducing pathways in cells of both the posterior and anterior eye in glaucoma. Despite intense research efforts over the past decades, much remains unknown about the mechanobiology of glaucoma. Continued investigation of glaucomatous mechanobiology is important, as it may reveal novel targets for treating this challenging disease.

Formation of transcellular pores facilitates material transport across endothelial barriers. In Schlemm’s canal (SC) endothelium, impaired pore formation is linked to glaucoma, but the underlying processes remain poorly understood due to a lack of in vitro assays. Here, we present a platform to study pore formation in human endothelial cells. Pores were induced in SC cells by seeding them atop micron-sized magnetic beads and applying a magnetic field to generate basal-to-apical forces, mimicking in vivo biomechanics. Pore formation was dynamic, with pores opening and closing. Glaucomatous cells showed impaired pore formation, which correlated with increased stiffness. Notably, apical-to-basal forces failed to induce pores in SC cells but triggered pore formation in other endothelial types. These findings highlight the central role of cell mechanics in transcellular pore formation and provide an approach to investigate the mechanisms underlying this process.

Pathological changes in the biomechanical environment of Schlemm's canal (SC) inner wall cells, such as substrate stiffening and increased cellular stretch, are associated with ocular hypertension, a key risk factor for the development of glaucoma. Cell membrane stretch can trigger the activation of transient receptor potential vanilloid 4 (TRPV4) mechanosensitive ion channels, allowing calcium influx and initiating downstream signaling. However, the precise role of TRPV4 in SC cell mechanobiology remains unclear. Here, we demonstrate that sustained inhibition of TRPV4 activity modulates substrate stiffness mechanosensing to thereby affect the remodeling of the actin cytoskeleton and extracellular matrix of SC cells. This is accompanied by a reduction in cell stiffness and an increase in transcellular pore forming ability, potentially lowering outflow resistance and risk of ocular hypertension. Interestingly, acute activation of TRPV4 channels induces Ca2+ influx, increasing transcellular pore formation in SC cells. Notably, reduced TRPV4 mechanosensing is observed in glaucomatous SC cells, resulting in reduced transcellular pore forming ability. These findings suggest novel potential strategies based on targeting TRPV4 in SC cells for the treatment of ocular hypertension in glaucoma. Statement of significance: This study identifies TRPV4 as a key mechanosensor in Schlemm's canal (SC) endothelial cells, modulating cytoskeletal dynamics, extracellular matrix remodeling, cell stiffness, and transcellular pore formation-all of which are processes central to intraocular pressure (IOP) regulation/determination. Although TRPV4 in other ocular tissues has been implicated in IOP modulation, its role in SC cell mechanobiology remained undefined. Using tissue-mimetic hydrogels and pharmacological modulation, we demonstrate that TRPV4 activity governs SC cell responses to substrate stiffness and affects transcellular pore forming ability. Notably, these mechanosensory functions are diminished in glaucomatous SC cells. Our findings underscore the complexity of SC cell mechanobiology in glaucoma and suggest that TRPV4-targeted interventions may need to be tailored to disease-specific cellular contexts.

Pathological changes in the biomechanical environment of Schlemm's canal (SC) inner wall cells, such as substrate stiffening and increased cellular stretch, are associated with ocular hypertension, a key risk factor for the development of glaucoma. Cell membrane stretch can trigger the activation of transient receptor potential vanilloid 4 (TRPV4) mechanosensitive ion channels, allowing calcium influx and initiating downstream signaling. However, the precise role of TRPV4 in SC cell mechanobiology remains unclear. Here, we demonstrate that sustained inhibition of TRPV4 activity modulates substrate stiffness mechanosensing to thereby affect the remodeling of the actin cytoskeleton and extracellular matrix of SC cells. This is accompanied by a reduction in cell stiffness and an increase in transcellular pore forming ability, potentially lowing outflow resistance and risk of ocular hypertension. Conversely, acute activation of TRPV4 channels induces Ca2+ influx, increasing transcellular pore formation in SC cells. Notaly, reduced TRPV4 mechanosensing was observed in glaucomatous SC cells, resulting in reduced transcellular pore forming ability. These findings suggest novel potential strategies based on targeting TRPV4 in SC cells for the treatment of ocular hypertension in glaucoma.

Purpose:Myopia incidence is increasing globally. All-trans retinoic acid (atRA) is important in myopigenic retinoscleral signaling, motivating research on its ocular transport. However, atRA's weak autofluorescence limits its direct visualization in tissues. Further, atRA is hydrophobic and must bind to protein carriers for transport. We assessed a fluorescent analog of atRA (LightOx 14, CAS:198696-03-6, referred as "floRA"), as an experimentally accessible atRA surrogate by: (i) evaluating its binding to carrier proteins and (ii) visualizing its distribution in ocular tissues. Methods:Binding: We assessed atRA-carrier protein binding using fluorescence quenching assays with bovine serum albumin (BSA), high density lipoprotein (HDL), apolipoprotein A-I (Apo A-I), and retinol binding protein 4 (RBP4). Direct visualization: Wild-type C57BL/6J mice were euthanized, their eyes were enucleated, and wedges containing sclera and choroid were incubated for specific durations in 50 µM floRA + BSA. The wedge centers were cryo-sectioned and counterstained for nuclei. Fluorescent micrographs were acquired and analyzed using ImageJ software. Results:Association constants (Ka) for atRA and floRA binding to carrier proteins were similar and ranged from 2 to 13 × 105 M-1, indicating nonspecific binding. floRA could be visualized in the sclera and choroid, yet showed significant spatial heterogeneity (enhanced fluorescence often colocalizing with nuclei). Conclusions:floRA is a reasonable surrogate for atRA binding to BSA, HDL, Apo A-I, and RBP4. Considering these proteins' relative serum and extravascular abundances, and their similar binding affinity to atRA, we predict that serum albumin is an important atRA carrier. Use of floRA in whole tissue tracer studies shows promise but requires further refinement.