Protective Effect of Nicotinamide Riboside on Glucocorticoid-Induced Glaucoma: Mitigating Mitochondrial Damage and Extracellular Matrix Deposition.

Purpose: Glucocorticoid-induced glaucoma (GIG) is a prevalent complication associated with glucocorticoids (GCs), resulting in irreversible blindness. GIG is characterized by the abnormal deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), elevation of intraocular pressure (IOP), and loss of retinal ganglion cells (RGCs). The objective of this study is to investigate the effects of nicotinamide riboside (NR) on TM in GIG. Methods: Primary human TM cells (pHTMs) and C57BL/6J mice responsive to GCs were utilized to establish in vitro and in vivo GIG models, respectively. The study assessed the expression of ECM-related proteins in TM and the functions of pHTMs to reflect the effects of NR. Mitochondrial morphology and function were also examined in the GIG cell model. GIG progression was monitored through IOP, RGCs, and mitochondrial morphology. Intracellular nicotinamide adenine dinucleotide (NAD+) levels of pHTMs were enzymatically assayed. Results: NR significantly prevented the expression of ECM-related proteins and alleviated dysfunction in pHTMs after dexamethasone treatment. Importantly, NR protected damaged ATP synthesis, preventing overexpression of mitochondrial reactive oxygen species (ROS), and also protect against decreased mitochondrial membrane potential induced by GCs in vitro. In the GIG mouse model, NR partially prevented the elevation of IOP and the loss of RGCs. Furthermore, NR effectively suppressed the excessive expression of ECM-associated proteins and mitigated mitochondrial damage in vivo. Conclusions: Based on the results, NR effectively enhances intracellular levels of NAD+, thereby mitigating abnormal ECM deposition and TM dysfunction in GIG by attenuating mitochondrial damage induced by GCs. Thus, NR has promising potential as a therapeutic candidate for GIG treatment.

Determining Isoprenoid-Facilitated Monomeric GTPase Turnover in Primary Human Trabecular Meshwork Cultures.

Members of the Rho family of small monomeric GTPases regulate a plethora of critical cellular functions including gene expression, cell cycle progression, and the dynamic modeling of the actin cytoskeleton. Diversity among Rho family members is derived, in part, from variations in their subcellular distribution. Localization of newly synthesized (naïve) Rho proteins to target subcellular compartments is largely governed by lipid modifications, including posttranslational prenylation. Here, using well-established and widely available contemporary methodologies, detailed protocols by which to semiquantitatively evaluate the functional consequence of posttranslational prenylation in human trabecular meshwork cells are described. We propose the novel concept that posttranslational prenylation itself is a key regulator of mammalian Rho GTPase protein expression and turnover.

Downregulation of ATP8B2 to Assess Plasmalogen Distribution and Far1 Expression in Primary Trabecular Meshwork Cells.

The trabecular meshwork (TM) from primary open-angle glaucoma (POAG) cases has been found to contain decreased levels of intracellular plasmalogens. Plasmalogens are a subset of lipids involved in diverse cellular processes such as intracellular signaling, membrane asymmetry, and protein regulation. Proper plasmalogen biosynthesis is regulated by rate-limiting enzyme fatty acyl-CoA reductase (Far1). ATPase phospholipid transporting 8B2 (ATP8B2) is a type IV P-type ATPase responsible for the asymmetric distribution of plasmalogens between the intracellular and extracellular leaflets of the plasma membranes. Here we describe the methodology for extraction and culturing of TM cells from corneal tissue and subsequent downregulation of ATP8B2 using siRNA transfection. Further quantification and downstream effects of ATP8B2 gene knockdown will be analyzed utilizing immunoblotting techniques.

The TGFß Induced MicroRNAome of the Trabecular Meshwork.

Primary open-angle glaucoma (POAG) is a progressive optic neuropathy with a complex, multifactorial aetiology. Raised intraocular pressure (IOP) is the most important clinically modifiable risk factor for POAG. All current pharmacological agents target aqueous humour dynamics to lower IOP. Newer therapeutic agents are required as some patients with POAG show a limited therapeutic response or develop ocular and systemic side effects to topical medication. Elevated IOP in POAG results from cellular and molecular changes in the trabecular meshwork driven by increased levels of transforming growth factor ß (TGFß) in the anterior segment of the eye. Understanding how TGFß affects both the structural and functional changes in the outflow pathway and IOP is required to develop new glaucoma therapies that target the molecular pathology in the trabecular meshwork. In this study, we evaluated the effects of TGF-ß1 and -ß2 treatment on miRNA expression in cultured human primary trabecular meshwork cells. Our findings are presented in terms of specific miRNAs (miRNA-centric), but given miRNAs work in networks to control cellular pathways and processes, a pathway-centric view of miRNA action is also reported. Evaluating TGFß-responsive miRNA expression in trabecular meshwork cells will further our understanding of the important pathways and changes involved in the pathogenesis of glaucoma and could lead to the development of miRNAs as new therapeutic modalities in glaucoma.

NAD+ supplementation improves mitochondrial functions and normalizes glaucomatous trabecular meshwork features.

Glaucoma is characterized by pathological elevation of intraocular pressure (IOP) due to dysfunctional trabecular meshwork (TM), which is the primary cause of irreversible vision loss. There are currently no effective treatment strategies for glaucoma. Mitochondrial function plays a crucial role in regulating IOP within the TM. In this study, primary TM cells treated with dexamethasone were used to simulate glaucomatous changes, showing abnormal cellular cytoskeleton, increased expression of extracellular matrix, and disrupted mitochondrial fusion and fission dynamics. Furthermore, glaucomatous TM cell line GTM3 exhibited impaired mitochondrial membrane potential and phagocytic function, accompanied by decreased oxidative respiratory levels as compared to normal TM cells iHTM. Mechanistically, lower NAD + levels in GTM3, possibly associated with increased expression of key enzymes CD38 and PARP1 related to NAD + consumption, were observed. Supplementation of NAD + restored mitochondrial function and cellular viability in GTM3 cells. Therefore, we propose that the aberrant mitochondrial function in glaucomatous TM cells may be attributed to increased NAD + consumption dependent on CD38 and PARP1, and NAD + supplementation could effectively ameliorate mitochondrial function and improve TM function, providing a novel alternative approach for glaucoma treatment.

Healing responses at the angle after micro-invasive glaucoma surgery-an AS-OCT study.

PURPOSE: To evaluate structural alterations and healing responses in the trabecular meshwork region with optical coherence tomography (AS-OCT) following after gonioscopy assisted transluminal trabeculotomy (GATT) and microincisional trabeculectomy (MIT). METHODS: 73 eyes of 67 patients (M:F = 45:22) with ≥6 months of follow-up after MIT (n = 41) or GATT (n = 32) with or without combined cataract surgery were included for this prospective study. The angle as seen on AS-OCT at 1, 3, 6 months after surgery were evaluated for structural alterations like peripheral anterior synechiae (PAS), hyphema, and hyperreflective scarring responses. The scarring was graded according to the linear extent measured from the centre of the trabecular meshwork (TM) gutter to the sclera/cornea as mild (<250µ), moderate (250-500µ), and severe(˃500µ), while the pattern of scarring was graded as open saucer/gutter, closed gutter, and trench pattern. The association of the need for medication or surgical outcome and clinical variables and AS-OCT parameters including the pattern and severity of scarring were analysed using multivariate regression. RESULTS: All eyes achieved significant reduction of IOP and number of medications with a final IOP of 15±3.2mm Hg at a mean follow-up of 8±32. months. While mild scarring was seen more common in MIT, severe scarring was seen in >65% of GATT eyes compared to 31% of MIT eye, p<0.001. An open saucer was equally seen in MIT and GATT while the trench pattern was more commonly seen in GATT eyes (>50%). Severe scarring in a trench pattern seemed to predict the need for medications for IOP control, though they independently did not seem to influence the final IOP or surgical outcome. CONCLUSION: A severe form of scarring in a trench pattern on AS-OCT predicted the need for glaucoma medications after MIGS surgery. Regular monitoring of the scarring responses by AS-OCT and clinical examination are necessary to identify those at need for medications after MIGS.

The Factors Affecting the Stability of IOP Homeostasis.

Purpose: 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). Methods: 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. Results: 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. Conclusions: 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.

Predictive biomarkers of intra-ocular pressure decrease after cataract surgery associated with trabecular washout in patients with pseudo exfoliative glaucoma.

To investigate biomarkers of intra-ocular pressure (IOP) decrease after cataract surgery with trabecular washout in pseudo-exfoliative (PEX) glaucoma. A single-center observational prospective study in PEX glaucoma patients undergoing cataract surgery with trabecular washout (Goniowash) was performed from 2018 to 2021. Age, gender, visual acuity, IOP, endothelial cell count, central corneal thickness, medications, were collected over 16-month follow-up. Multivariable binomial regression models were implemented. 54 eyes (35 subjects) were included. Mean preoperative IOP (IOPBL) was 15.9 ± 3.5 mmHg. Postoperative IOP reduction was significant at 1-month and throughout follow-up (p < 0.01, respectively). IOPBL was a predictive biomarker inversely correlated to IOP decrease throughout follow-up (p < 0.001). At 1 and 12 months of follow-up, IOP decrease concerned 31 (57.4%) and 34 (63.0%) eyes with an average IOP decrease of 17.5% (from 17.6 ± 3.1 to 14.3 ± 2.2 mmHg) and 23.0% (from 17.7 ± 2.8 to 13.5 ± 2.6 mmHg), respectively. Performance (AUC) of IOPBL was 0.85 and 0.94 (p < 0.0001, respectively), with IOPBL threshold ≥ 15 mmHg for 82.1% and 96.8% sensitivity, 84.2% and 75.0% specificity, 1.84 and 3.91 IOP decrease odds-ratio, respectively. All PEX glaucoma patients with IOPBL greater than or equal to the average general population IOP were likely to achieve a significant sustainable postoperative IOP decrease.

DDX58 variant triggers IFN-ß-induced autophagy in trabecular meshwork and influences intraocular pressure.

Singleton-Merten syndrome (SMS) is a rare immunogenetic disorder affecting multiple systems, characterized by dental dysplasia, aortic calcification, glaucoma, skeletal abnormalities, and psoriasis. Glaucoma, a key feature of both classical and atypical SMS, remains poorly understood in terms of its molecular mechanism caused by DDX58 mutation. This study presented a novel DDX58 variant (c.1649A>C [p.Asp550Ala]) in a family with childhood glaucoma. Functional analysis showed that DDX58 variant caused an increase in IFN-stimulated gene expression and high IFN-ß-based type-I IFN. As the trabecular meshwork (TM) is responsible for controlling intraocular pressure (IOP), we examine the effect of IFN-ß on TM cells. Our study is the first to demonstrate that IFN-ß significantly reduced TM cell viability and function by activating autophagy. In addition, anterior chamber injection of IFN-ß remarkably increased IOP level in mice, which can be attenuated by treatments with autophagy inhibitor chloroquine. To uncover the specific mechanism underlying IFN-ß-induced autophagy in TM cells, we performed microarray analysis in IFN-ß-treated and DDX58 p.Asp550Ala TM cells. It showed that RSAD2 is necessary for IFN-ß-induced autophagy. Knockdown of RSAD2 by siRNA significantly decreased autophagy flux induced by IFN-ß. Our findings suggest that DDX58 mutation leads to the overproduction of IFN-ß, which elevates IOP by modulating autophagy through RSAD2 in TM cells.

Silibinin attenuates TGF-ß2-induced fibrogenic changes in human trabecular meshwork cells by targeting JAK2/STAT3 and PI3K/AKT signaling pathways.

Transforming growth factor-ß2 (TGF-ß2) induced fibrogenic changes in human trabecular meshwork (HTM) cells have been implicated in trabecular meshwork (TM) damage and intraocular pressure (IOP) elevation in primary open-angle glaucoma (POAG) patients. Silibinin (SIL) exhibited anti-fibrotic properties in various organs and tissues. This study aimed to assess the effects of SIL on the TGF-ß2-treated HTM cells and to elucidate the underlying mechanisms. Our study found that SIL effectively inhibited HTM cell proliferation, attenuated TGF-ß2-induced cell migration, and mitigated TGF-ß2-induced reorganization of both actin and vimentin filaments. Moreover, SIL suppressed the expressions of fibronectin (FN), collagen type I alpha 1 chain (COL1A1), and alpha-smooth muscle actin (α-SMA) in the TGF-ß2-treated HTM cells. RNA sequencing indicated that SIL interfered with the phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as AKT) signaling pathway, extracellular matrix (ECM)-receptor interaction, and focal adhesion in the TGF-ß2-treated HTM cells. Western blotting demonstrated SIL inhibited the activation of Janus kinase 2 (JAK2)/signal transducers and activators of transcription 3 (STAT3) and the downstream PI3K/AKT signaling pathways induced by TGF-ß2, potentially contributing to its inhibitory effects on ECM protein production in the TGF-ß2-treated HTM cells. Our study demonstrated the ability of SIL to inhibit TGF-ß2-induced fibrogenic changes in HTM cells. SIL could be a potential IOP-lowering agent by reducing the fibrotic changes in the TM tissue of POAG patients, which warrants further investigation through additional animal and clinical studies.

SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma.

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients' surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Profiling IOP-Responsive Genes in the Trabecular Meshwork and Optic Nerve Head in a Rat Model of Controlled Elevation of Intraocular Pressure.

Purpose: The rat controlled elevation of intraocular pressure (CEI) model allows study of in vivo responses to short-term exposure to defined intraocular pressures (IOP). In this study, we used NanoString technology to investigate in vivo IOP-related gene responses in the trabecular meshwork (TM) and optic nerve head (ONH) simultaneously from the same animals. Methods: Male and female rats (N = 35) were subjected to CEI for 8 hours at pressures simulating mean, daytime normotensive rat IOP (CEI-20), or 2.5× IOP (CEI-50). Naïve animals that received no anesthesia or surgical interventions served as controls. Immediately after CEI, TM and ONH tissues were dissected, RNA was isolated, and samples were analyzed with a NanoString panel containing 770 genes. Postprocessing, raw count data were uploaded to ROSALIND for differential gene expression analyses. Results: For the TM, 45 IOP-related genes were significant in the CEI-50 versus CEI-20 and CEI-50 versus naïve comparisons, with 15 genes common to both comparisons. Bioinformatics analysis identified Notch and transforming growth factor beta (TGFß) pathways to be the most up- and downregulated Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, respectively. For ONH, 22 significantly differentially regulated genes were identified in the CEI-50 versus naïve comparison. Pathway analysis identified defense response and immune response as two significantly upregulated biological process pathways. Conclusions: This study demonstrated the ability to assay short-term IOP-responsive genes in both TM and ONH tissues simultaneously. In the TM, downregulation of TGFß pathway genes suggests that TM responses may reduce TGFß-induced extracellular matrix synthesis. For ONH, the initial response to short-term elevated IOP may be protective.

[To discuss the minimally invasive glaucoma surgery related to trabeculotomy from the perspective of physiological function of trabecular meshwork drainage pathway].

The advent of minimally invasive glaucoma surgery (MIGS) has broadened the therapeutic options for managing glaucoma. In recent years, MIGS procedures targeting the trabecular meshwork-Schlemm's canal aqueous outflow resistance site have garnered significant attention. This focus has extended to the pathophysiological changes occurring within the aqueous outflow pathway. However, questions persist regarding the efficacy of near-peripheral or peripheral trabeculotomy in achieving the anticipated reduction of outflow resistance and the suitability of MIGS surgery for patients with primary open-angle glaucoma. By integrating clinical experience with pertinent clinical research, this paper advocates for a reevaluation of MIGS procedures to aid clinicians in making informed decisions regarding various glaucoma surgical interventions.

Paeoniflorin alleviates TGF-ß2-mediated extracellular matrix remodeling and oxidative stress in human trabecular meshwork cells.

BACKGROUND: The multifunctional profibrotic cytokine transforming growth factor-beta2 (TGF-ß2) is implicated in the pathophysiology of primary open angle glaucoma. Paeoniflorin (PAE) is a monoterpene glycoside with multiple pharmacological efficacies, such as antioxidant, anti-fibrotic, and anti-inflammatory properties. Studies have demonstrated that paeoniflorin protects human corneal epithelial cells, retinal pigment epithelial cells, and retinal microglia from damage. Here, the biological role of PAE in TGF-ß2-dependent remodeling of the extracellular matrix (ECM) within the trabecular meshwork (TM) microenvironment. METHODS: Primary or transformed (GTM3) human TM (HTM) cells conditioned in serum-free media were incubated with TGF-ß2 (5 ng/mL). PAE (300 µM) was added to serum-starved confluent cultures of HTM cells for 2 h, followed by incubation with TGF-ß2 for 22 h. SB-431542, a TGF-ß receptor inhibitor (10 µM), was used as a positive control. The levels of intracellular ROS were evaluated by CellROX green dye. Western blotting was used to measure the levels of TGF-ß2/Smad2/3 signaling-related molecules. Collagen 1α1, collagen 4α1, and connective tissue growth factor (CTGF) expression was evaluated by RT-qPCR. Immunofluorescence assay was conducted to measure collagen I/IV expression in HTM cells. Phalloidin staining assay was conducted for evaluating F-actin stress fiber formation in the cells. RESULTS: PAE attenuated TGF-ß2-induced oxidative stress and suppressed TGF-ß2-induced Smad2/3 signaling in primary or transformed HTM cells. Additionally, PAE repressed TGF-ß2-induced upregulation of collagen 1α1, collagen 4α1, and CTGF expression and reduced TGF-ß2-mediated collagen I/IV expression and of F-actin stress fiber formation in primary or transformed HTM cells. CONCLUSION: PAE alleviates TGF-ß2-induced ECM deposition and oxidative stress in HTM cells through inactivation of Smad2/3 signaling.

Clinical applications of aqueous angiography in glaucoma.

Aqueous humor outflow (AHO) pathways are the main site of resistance causing elevated intraocular pressure in glaucoma, especially primary open-angle glaucoma patients. With the recently introduced technique of aqueous angiography (AA); functional, real time assessment of AHO from proximal (trabecuar meshwork) to distal pathways under physiological conditions has been made possible. AHO pathways are segmental, and AA can identify high-flow region (increased angiographic signals) and low flow region (decreased angiographic signals) in an individual. With the introduction of canal-based minimally invasive glaucoma surgeries (MIGS), the assessment of AHO can help guide the placement of stents/incisions during MIGS procedures. This can allow individualized and targeted MIGS procedures in glaucoma patients for better results. Based on the density of AHO pathways visualized on AA, surgeons can decide whether to perform MIGS or conventional glaucoma surgery for improved outcomes for the patient. Immediate intraoperative assessment for functionality of the MIGS procedure performed is possible with AA, allowing for surgical adjustments of MIGS procedure in the same sitting, if needed. This review provides a summary of the studies performed with AA to date, with a special focus on Indian patients. It covers the basics and clinical applications of AA for improving surgical outcomes in glaucoma patients.

Development of Cell Culture Platforms for Study of Trabecular Meshwork Cells and Glaucoma Development.

BACKGROUND: Various cell culture platforms that could display native environmental cue-mimicking stimuli were developed, and effects of environmental cues on cell behaviors were studied with the cell culture platforms. Likewise, various cell culture platforms mimicking native trabecular meshwork (TM) composed of juxtacanalicular, corneoscleral and uveal meshwork located in internal scleral sulcus were used to study effects of environmental cues and/or drug treatments on TM cells and glaucoma development. Glaucoma is a disease that could cause blindness, and cause of glaucoma is not clearly identified yet. It appears that aqueous humor (AH) outflow resistance increased by damages on pathway of AH outflow can elevate intraocular pressure (IOP). These overall possibly contribute to development of glaucoma. METHODS: For the study of glaucoma, static and dynamic cell culture platforms were developed. Particularly, the dynamic platforms exploiting AH outflow-mimicking perfusion or increased IOP-mimicking increased pressure were used to study how perfusion or increased pressure could affect TM cells. Overall, potential mechanisms of glaucoma development, TM structures and compositions, TM cell culture platform types and researches on TM cells and glaucoma development with the platforms were described in this review. RESULTS AND CONCLUSION: This will be useful to improve researches on TM cells and develop enhanced therapies targeting glaucoma.

Aqueous humor as eye lymph: A crossroad between venous and lymphatic system.

Aqueous humor (AQH) is a transparent fluid with characteristics similar to those of the interstitial fluid, which fills the eyeball posterior and anterior chambers and circulates in them from the sites of production to those of drainage. The AQH volume and pressure homeostasis is essential for the trophism of the ocular avascular tissues and their normal structure and function. Different AQH outflow pathways exist, including a main pathway, quite well defined anatomically and referred to as the conventional pathway, and some accessory pathways, more recently described and still not fully morphofunctionally understood, generically referred to as unconventional pathways. The conventional pathway is based on the existence of a series of conduits starting with the trabecular meshwork and Schlemm's Canal and continuing with a system of intrascleral and episcleral venules, which are tributaries to veins of the anterior segment of the eyeball. The unconventional pathways are mainly represented by the uveoscleral pathway, in which AQH flows through clefts, interstitial conduits located in the ciliary body and sclera, and then merges into the aforementioned intrascleral and episcleral venules. A further unconventional pathway, the lymphatic pathway, has been supported by the demonstration of lymphatic microvessels in the limbal sclera and, possibly, in the uvea (ciliary body, choroid) as well as by the ocular glymphatic channels, present in the neural retina and optic nerve. It follows that AQH may be drained from the eyeball through blood vessels (TM-SC pathway, US pathway) or lymphatic vessels (lymphatic pathway), and the different pathways may integrate or compensate for each other, optimizing the AQH drainage. The present review aims to define the state-of-the-art concerning the structural organization and the functional anatomy of all the AQH outflow pathways. Particular attention is paid to examining the regulatory mechanisms active in each of them. The new data on the anatomy and physiology of AQH outflow pathways is the key to understanding the pathophysiology of AQH outflow disorders and could open the way for novel approaches to their treatment.

Comparative analysis of traction forces in normal and glaucomatous trabecular meshwork cells within a 3D, active fluid-structure interaction culture environment.

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.5 and 21.7 kPa, is based on serial block-face scanning electron microscopy images of the outflow pathway. It allows for quantifying 3D, depth-dependent, dynamic traction forces exerted by both normal and glaucomatous TM cells within an active fluid-structure interaction (FSI) environment. In our experimental design, we designed two scenarios: a control group with TM cells observed over 20 hours without flow (static setting), focusing on intrinsic cellular contractile forces, and a second scenario incorporating active FSI to evaluate its impact on traction forces (dynamic setting). Our observations revealed that active FSI results in higher traction forces (normal: 1.83-fold and glaucoma: 2.24-fold) and shear strains (normal: 1.81-fold and glaucoma: 2.41-fold), with stiffer substrates amplifying this effect. Glaucomatous cells consistently exhibited larger forces than normal cells. Increasing gel stiffness led to enhanced stress fiber formation in TM cells, particularly in glaucomatous cells. Exposure to active FSI dramatically altered actin organization in both normal and glaucomatous TM cells, particularly affecting cortical actin stress fiber arrangement. This model while preliminary offers a new method in understanding TM cell biomechanics and ECM stiffening in glaucoma, highlighting the importance of FSI in these processes. STATEMENT OF SIGNIFICANCE: This pioneering project presents an advanced 3D in vitro model, meticulously replicating the human trabecular meshwork's anatomy for glaucoma research. It enables precise quantification of cellular forces in a dynamic fluid-structure interaction, a leap forward from existing 2D models. This advancement promises significant insights into trabecular meshwork cell biomechanics and the stiffening of the extracellular matrix in glaucoma, offering potential pathways for innovative treatments. This research is positioned at the forefront of ocular disease study, with implications that extend to broader biomedical applications.

Microstructure of the corneal endothelial transition zone in different laboratory animals.

Purpose: To compare the microstructure of the corneal endothelial transition zone in different laboratory animals. Methods: Flat-mount corneas of rabbits, rats, and mice were stained with Alizarin Red S (ARS) and observed using scanning electron microscopy (SEM). The progenitor cell markers p75 neurotrophin receptor (p75NTR), SRY-box transcription factor 9 (SOX9), leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5), telomerase reverse transcriptase (TERT), and proliferation marker Ki-67 were examined in the flat-mounted corneas of three laboratory animals using immunofluorescence microscopy. Results: On flat mounts, proximity to the trabecular meshwork correlated with weaker ARS staining and greater polymorphism of endothelial cells in the transition zone in all animals. On SEM, distinct and smooth structures of the transition zone were negligibly detected in all animals. The endothelial cells in the transition zone had irregular shapes, with less dense, less wavy intercellular junctions, especially in murine corneas, exhibiting unique intercellular cystic spaces. In the transition zone of the rabbit cornea, progenitor cell markers p75NTR, SOX9, Lgr5, TERT, and proliferation marker Ki-67 were expressed, in contrast to those in other murine corneas. Conclusions: Although the transition zone was not identified clearly, irregular cell morphology and loss of cell-cell contact were observed in all animal corneal endothelial cells. The proliferative capacity and the presence of progenitor cells were confirmed in the transition zone, especially in the rabbit cornea.

A digoxin derivative that potently reduces intraocular pressure: efficacy and mechanism of action in different animal models.

Glaucoma is a blinding disease. Reduction of intraocular pressure (IOP) is the mainstay of treatment, but current drugs show side effects or become progressively ineffective, highlighting the need for novel compounds. We have synthesized a family of perhydro-1,4-oxazepine derivatives of digoxin, the selective inhibitor of Na,K-ATPase. The cyclobutyl derivative (DcB) displays strong selectivity for the human α2 isoform and potently reduces IOP in rabbits. These observations appeared consistent with a hypothesis that in ciliary epithelium DcB inhibits the α2 isoform of Na,K-ATPase, which is expressed strongly in nonpigmented cells, reducing aqueous humor (AH) inflow. This paper extends assessment of efficacy and mechanism of action of DcB using an ocular hypertensive nonhuman primate model (OHT-NHP) (Macaca fascicularis). In OHT-NHP, DcB potently lowers IOP, in both acute (24 h) and extended (7-10 days) settings, accompanied by increased aqueous humor flow rate (AFR). By contrast, ocular normotensive animals (ONT-NHP) are poorly responsive to DcB, if at all. The mechanism of action of DcB has been analyzed using isolated porcine ciliary epithelium and perfused enucleated eyes to study AH inflow and AH outflow facility, respectively. 1) DcB significantly stimulates AH inflow although prior addition of 8-Br-cAMP, which raises AH inflow, precludes additional effects of DcB. 2) DcB significantly increases AH outflow facility via the trabecular meshwork (TM). Taken together, the data indicate that the original hypothesis on the mechanism of action must be revised. In the OHT-NHP, and presumably other species, DcB lowers IOP by increasing AH outflow facility rather than by decreasing AH inflow.NEW & NOTEWORTHY When applied topically, a cyclobutyl derivative of digoxin (DcB) potently reduces intraocular pressure in an ocular hypertensive nonhuman primate model (Macaca fascicularis), associated with increased aqueous humor (AH) flow rate (AFR). The mechanism of action of DcB involves increased AH outflow facility as detected in enucleated perfused porcine eyes and, in parallel, increased (AH) inflow as detected in isolated porcine ciliary epithelium. DcB might have potential as a drug for the treatment of open-angle human glaucoma.