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‘What controls aqueous humour outflow resistance?’
Abstract
The bulk of aqueous humour outflow resistance is generated in or near the inner wall endothelium of Schlemm's canal in normal eyes, and probably also in glaucomatous eyes. Fluid flow through this region is controlled by the location of the giant vacuoles and pores found in cells of the endothelium of Schlemm's canal, but the flow resistance itself is more likely generated either in the extracellular matrix of the juxtacanalicular connective tissue or the basement membrane of Schlemm's canal. Future studies utilizing in vitro perfusion studies of inner wall endothelial cells may give insights into the process by which vacuoles and pores form in this unique endothelium and why inner wall pore density is greatly reduced in glaucoma.
... In some cases, the outflow of aqueous humor can become obstructed, leading to an increase in IOP. 42 Some of the processes involving the dynamics of the tear film, aqueous humour, and vitreous humour under various conditions have been studied by mathematicians, physicists, and engineers over many years. [43][44][45][46][47] However, similar comprehensive works in the context of non-contact tonometry are relatively sparse. ...
... The aqueous humor is a clear, watery fluid that fills the space between the cornea and the eye's lens. 41,42 The wave generated by the air puff propagates through the aqueous humor and interacts with the eye's lens. This interaction causes the lens to move slightly, which can affect the measurement of the IOP. ...
Non-contact tonometry (NCT) is a non-invasive ophthalmologic technique to measure intraocular pressure (IOP) using an air puff for routine glaucoma testing. Although IOP measurement using NCT has been perfected over many years, various phenomenological aspects of interfacial physics, fluid structure interaction, waves on corneal surface, and pathogen transmission routes to name a few are inherently unexplored. Research investigating the interdisciplinary physics of the ocular biointerface and of the NCT procedure is sparse and hence remains to be explored in sufficient depth. In this perspective piece, we introduce NCT and propose future research prospects that can be undertaken for a better understanding of the various hydrodynamic processes that occur during NCT from a pathogen transmission viewpoint. In particular, the research directions include the characterization and measurement of the incoming air puff, understanding the complex fluid-solid interactions occurring between the air puff and the human eye for measuring IOP, investigating the various waves that form and travel; tear film breakup and subsequent droplet formation mechanisms at various spatiotemporal length scales. Further, from an ocular disease transmission perspective, the disintegration of the tear film into droplets and aerosols poses a potential pathogen transmission route during NCT for pathogens residing in nasolacrimal and nasopharynx pathways. Adequate precautions by opthalmologist and medical practioners are therefore necessary to conduct the IOP measurements in a clinically safer way to prevent the risk associated with pathogen transmission from ocular diseases like conjunctivitis, keratitis, and COVID-19 during the NCT procedure.
... Der individuell zu hohe Intraokulardruck (IOD) ist der Hauptrisikofaktor für die Entstehung eines Glaukoms [2]. Dieser wird hauptsächlich durch einen erhöhten Abflusswiderstand des Kammerwassers im juxtakanalikulären Trabekelmaschenwerk und an der anliegenden inneren Wand des Schlemm-Kanals herbeigeführt [3]. Randomisierte, kontrollierte Studien haben bewiesen, dass durch die Senkung des IOD eine Verringerung des glaukomatösen Schadens an den Sehnervenfasern erreicht werden kann [4,5]. ...
... Um eine belastbare Grundlage für die Beurteilung zu erhalten, ob bei Patienten, die im Laufe der 5 postoperativen Jahre nicht mehr zur Kontrolle zu uns kamen, wesentlich andere Ergebnisverläufe bestehen als bei den hier kontrollierten Patienten, wurden diese nach Abschluss der Studie nochmals kontaktiert und befragt, ob bei ihnen seit der letzten Kontrolle bei uns ein weiterer operativer drucksenkender Eingriff erforderlich geworden sei, ob seither eine Verstärkung der medikamentösen topischen Therapie erforderlich Die Ophthalmologie 3 Abb. 2 9 Kumulative Erfolgsraten des Intraokulardrucks nach TOT und PIT mit Erfolgskriterien 1 und 2 mit drucksenkenden Medikamenten ("qualified success") Abb. 3 Von den ursprünglich eingeschlossenen Patienten konnten 66 von 90 Patienten in der Gruppe der TOT und 22 von 31 Patienten in der Gruppe der PIT bis zum letzten Kontrolltermin nach 5 Jahren nachuntersucht werden. Nach Jahresintervallen waren dies in den jeweiligen Gruppen (TOT/PIT) nach 1 Jahr 95,6 %/96,8 %, nach 2 Jahren 87,8 %/80,6 %, nach 3 Jahren 82,2 %/83,9 %,nach4 Jahren75,6 %/83,9 % und nach 5 Jahren 73,3 %/71,0 %. ...
The published results of trabeculotomy ab externo (TOT), as an outflow channel surgery in adults with open-angle glaucoma, are underrepresented in comparison with other pressure-lowering glaucoma procedures.
To determine the long-term pressure lowering effect and complications of TOT as a stand-alone procedure or in combination with phacoemulsification with intraocular lens implantation (PIT) as a primary pressure-lowering procedure in adult patients with forms of chronic open-angle glaucoma.
This was a retrospective analysis of 121 eyes of 106 patients wherby 90 eyes underwent TOT and 31 eyes underwent PIT. The follow-up period was 5 years. Follow-up was performed in the same center under standardized conditions. Preoperative as well as postoperative best-corrected distance visual acuity, intraocular pressure, pressure-lowering medication, and complications were recorded. For success criterion 1, the intraocular pressure was not allowed to exceed 17 mm Hg at any control. For success criterion 2, the intraocular pressure was allowed to be above 17 mm Hg at 1 control throughout the follow-up period.
Preoperative intraocular pressure in the TOT group was 25.92 mm Hg (SD 6.58) and 26.32 mm Hg (SD 6.06) in the PIT group. The annual cumulative success rates with pressure-lowering medications for success criterion 1 were 92.8%, 82.5%, 70.5%, 52.7% and 26.1%. For success criterion 2 these were 97.9%; 94.9%, 93.2%, 91.3%, and 89%. We found no relevant difference in pressure reduction between TOT and PIT. Without pressure-lowering medications, success rates were significantly lower for both success criteria. No serious complications occurred. The most common postoperative complication was spontaneously resorbable hyphema.
Performing TOT or PIT as a primary glaucoma procedure for chronic open-angle glaucoma in adults is reasonable, has a significant pressure-lowering effect and very low complication rate.
... In the TM, aqueous humor flows through the intertrabecular spaces between TMBs before passing through pores in the inner wall of the AP, disrupting the laminar flow and reducing shear stress. Additionally, compared to CCs and ESVs, the TM and AP have a larger open space or diameter and lower resistance (Johnson, 2006;Gong and Francis, 2014). Hence, aqueous humor may experience less turbulence and Glycocalyx distribution and thickness in high-flow and low-flow regions along the trabecular outflow pathway. ...
Background: Glycocalyx is a hair-like structure covering the endothelium
of the aqueous outflow pathway. While trabecular outflow is segmental
circumferentially around the eye, regional differences in glycocalyx morphology
remain largely unexplored. This study investigated glycocalyx variations in the
different structures along the trabecular outflow pathway in high-flow (HF) and
low-flow (LF) regions of bovine eyes.
Methods: Enucleated bovine eyes (n = 8) were perfused with fluorescein to
identify HF and LF regions. The glycocalyx was labeled with Alcian Blue 8GX,
and radial wedges from the anterior chamber angles of both HF and LF regions
were processed for transmission electron microscopy. Glycocalyx thickness
and coverage were quantified using ImageJ and compared between different
outflow pathway locations in HF and LF regions. Glycocalyx measurements at
intracellular (I-pores) and border pores (B-pores), the percentage of glycocalyxunfilled
pores, as well as the percentage of giant vacuoles (GVs) with and without
I-pores with glycocalyx lining the inner membrane were evaluated.
Results: Glycocalyx thickness and coverage did not differ significantly between
HF and LF regions. However, thickness progressively increased from the proximal
(trabecular meshwork) to the distal (episcleral veins) outflow pathway. In both Ipores
and B-pores, the glycocalyx was present near the basal opening, edge, and
center of the pores, with thickness increasing toward the center. No significant
differences in the percentage of glycocalyx-filled pores were observed between
HF and LF regions. However, the percentage of GVs with I-pores exhibiting
glycocalyx lining the inner cellular membrane was significantly higher (100%)
than that of those without I-pores (16%).
Conclusion: No regional differences were found between HF and LF
regions, but glycocalyx thickness progressively increased from the proximal
to the distal outflow pathway, potentially reflecting varying shear stress
conditions. The significantly higher percentage of GVs with I-pores containing
glycocalyx lining the inner cellular membrane compared to those without
I-pores suggests a relationship between aqueous outflow dynamics and
glycocalyx synthesis. These findings provide a morphological basis for future research on glycocalyx alterations in glaucoma and their impact on outflow
resistance.
... 6 In the trabecular pathways, aqueous flows from the anterior chamber to the trabecular meshwork (TM), through Schlemm's canal (SC), and then into distal outflow pathways, including collector channels, the intracellular venous plexus, and aqueous/episcleral veins before returning aqueous to the blood circulation. 7,8 However, this traditional linear description does not fully describe AHO circumferentially around the limbus in a three-dimensional (3D) eye. ...
Purpose
To use robotic visible-light optical coherence tomography (vis-OCT) to study circumferential segmental Schlemm's canal (SC) anatomy in mice after topical pilocarpine administration.
Methods
Anterior segment imaging using a robotic vis-OCT to maintain perpendicular laser illumination aimed at SC was performed. Sixteen mice were studied for repeatability testing and to study aqueous humor outflow (AHO) pathway response to topical drug. Pharmaceutical-grade pilocarpine (1%; n = 5) or control artificial tears (n = 9) were given, and vis-OCT imaging was performed before and 15 minutes after drug application. SC areas and volumes were measured circumferentially.
Results
Circumferential vis-OCT provided high-resolution imaging of the AHO pathways. Segmental SC anatomy was visualized with the average cross-sectional area greatest temporal (3971 ± 328 µm²) and the least nasal (2727 ± 218 µm²; P = 0.018). After pilocarpine administration, the SC became larger (pilocarpine, 26.8 ± 5.0% vs. control, 8.9 ± 4.6% volume increase; P = 0.030). However, the pilocarpine alteration was also segmental, with a greater increase observed superior (pilocarpine, 31.6 ± 8.9% vs. control, 1.8 ± 5.7% volume increase; P = 0.023) and nasal (pilocarpine, 41.1 ± 15.3% vs. control, 13.9 ± 4.5% volume increase; P = 0.045).
Conclusions
Circumferential noninvasive imaging of the AHO pathways was done in vivo. Segmental SC anatomy was seen, consistent with the known segmental nature of trabecular AHO. Segmental SC anatomical response to a muscarinic agonist was also observed. Segmental glaucoma drug response around the circumference of AHO pathways is an observation that may influence patient response to glaucoma treatments.
... In addition to maintaining a barrier, the cell junctions also serve as an extracellular outflow route through pores at the cell border (Bpores) 3,9,13,14 . Flow also occurs through a transcellular route where pressure-modulated intracellular pores (I-pores) in giant vacuoles form in the inner wall SECs to allow AQH outflow 9,15 . ...
Schlemm’s canal endothelial cells (SECs) serve as the final barrier to aqueous humor (AQH) drainage from the eye. SECs adjust permeability to AQH outflow to modulate intraocular pressure (IOP). The broad identification of IOP-related genes implicates SECs in glaucoma. However, the molecular mechanisms by which SECs sense and respond to pressure changes to regulate fluid permeability and IOP remain largely undefined. We hypothesize that mechano-responsive phosphorylation of the cell adhesion molecule VE-CADHERIN (CDH5) in SECs, by FYN and possibly other SRC family kinases, regulates adherens junction (AJ) permeability to AQH in response to IOP. On experimentally raising IOP in mouse eyes, AJ permeability, CDH5 phosphorylation, and FYN activation at the AJ all increase. FYN null mutant mice display disrupted IOP regulation and reduced AQH outflow. These findings demonstrate an important role of mechanotransducive signaling within SECs in maintaining IOP homeostasis and implicate FYN as a potential target for developing IOP-lowering treatments.
... 12 Most of the conventional outflow resistance resides in the deepest part of the TM and SCE, but a significant portion is also located beyond Schlemm's canal. 4,9,10,[13][14][15][16][17][18] In the early 1980s, Polansky and colleagues [19][20][21][22] were successful in establishing primary cultures of TM cells, and, in 1998, Stamer and colleagues 23 isolated SCE cells. This allowed for biochemical and molecular analysis of these cultured cells. ...
Intraocular pressure (IOP) elevation is the primary risk factor and currently the main treatable factor for progression of glaucomatous optic neuropathy. In addition to direct clinical and living animal in vivo studies, ex vivo perfusion of anterior segments and whole eyes is a key technique for studying conventional outflow function as it is responsible for IOP regulation. We present well-tested experimental details, protocols, considerations, advantages, and limitations of several ex vivo model systems for studying IOP regulation. These include: (1) perfused whole globes, (2) stationary anterior segment organ culture, (3) perfused human anterior segment organ culture, (4) perfused animal anterior segment organ culture, (5) perfused human corneal rims, and (6) perfused human anterior segment wedges. These methods, with due consideration paid to their strengths and limitations, comprise a set of very strong tools for extending our understanding of IOP regulation.
... In vivo, a proportion of TM1 cells have a shorter, more spherical nuclear morphology, consistent with JCT cell identity. Importantly, the JCT region is critical in determining resistance to AH outflow and in regulating IOP (Acott and Kelley, 2008;Ethier, 2002;Johnson, 2006;Stamer and Acott, 2012). JCT cells have fibroblast-like properties, including the secretion of ECM proteins and degradation enzymes to support continuous ECM remodeling (Acott et al., 1988;Keller et al., 2009;Stamer and Clark, 2017). ...
Since the trabecular meshwork (TM) is central to intraocular pressure (IOP) regulation and glaucoma, a deeper understanding of its genomic landscape is needed. We present a multimodal, single-cell resolution analysis of mouse limbal cells (includes TM). In total, we sequenced 9,394 wild-type TM cell transcriptomes. We discovered three TM cell subtypes with characteristic signature genes validated by immunofluorescence on tissue sections and whole-mounts. The subtypes are robust, being detected in datasets for two diverse mouse strains and in independent data from two institutions. Results show compartmentalized enrichment of critical pathways in specific TM cell subtypes. Distinctive signatures include increased expression of genes responsible for 1) extracellular matrix structure and metabolism (TM1 subtype), 2) secreted ligand signaling to support Schlemm’s canal cells (TM2), and 3) contractile and mitochondrial/metabolic activity (TM3). ATAC-sequencing data identified active transcription factors in TM cells, including LMX1B. Mutations in LMX1B cause high IOP and glaucoma. LMX1B is emerging as a key transcription factor for normal mitochondrial function and its expression is much higher in TM3 cells than other limbal cells. To understand the role of LMX1B in TM function and glaucoma, we single-cell sequenced limbal cells from Lmx1b V265D/+ mutant mice. In V265D/+ mice, TM3 cells were uniquely affected by pronounced mitochondrial pathway changes. This supports a primary role of mitochondrial dysfunction within TM3 cells in initiating the IOP elevation that causes glaucoma in these mice. Importantly, treatment with vitamin B 3 (nicotinamide), to enhance mitochondrial function and metabolic resilience, significantly protected Lmx1b mutant mice from IOP elevation.
... 14,15 All existing medical and surgical treatments for glaucoma aim to lower intraocular pressure (IOP) to decelerate disease progression; 16,17 yet, existing approaches are insufficient. 18 IOP is strongly influenced by AH transport through micronsized pores that form in endothelial cells lining the inner wall of SC, the only continuous cellular barrier within the major pathway for drainage of AH from the eye 19 (Fig. 1a). Recent discoveries showing that SC pore formation is a mechanosensitive process and that pore formation can be facilitated by mechanical stretching 20 and transendothelial perfusion, 21 motivate the development of discovery tools to study the mechanism(s) of mechanosensitive pore formation. ...
Formation of transcellular pores facilitates the transport of materials across endothelial barriers. In Schlemm's canal (SC) endothelium, impaired pore formation is associated with glaucoma. However, our understanding of the cellular processes responsible for pore formation is limited by lack of in vitro assays. Here, we present a novel platform for studying transcellular pore formation in human endothelial cells. We induced pores in SC cells by seeding them atop micron-sized magnetic beads followed by application of a magnetic field to subject cells to a basal to apical force, mimicking in vivo biomechanical forces. The pore formation process was dynamic, with pores opening and closing. Glaucomatous cells exhibited impaired pore formation that correlated with their increased stiffness. We further discovered that application of forces from the apical to basal direction did not induce pores in SC cells but resulted in formation of pores in other types of endothelial cells. Our studies reveal the central role of cell mechanics in formation of transcellular pores in endothelial cells, and provide a new approach for investigating their associated underlying mechanism/s.
... aqueous humour across the uvea 8 . The generation and regulation of the IOP is mostly depended on the trabecular pathway, where the outflow resistance is localized in the inner wall region containing the juxtacanalicular tissue of the TM as well as the inner wall endothelium of Schlemm's canal 9 . ...
The trabecular meshwork (TM) is crucial for regulating intraocular pressure (IOP), and its dysfunction significantly contributes to glaucoma, a leading cause of vision loss and blindness worldwide. Although rodents are commonly used as animal models in glaucoma research, the applicability of these findings to humans is limited due to the insufficient understanding of murine TM. This study aimed to compare primary human TM (hTM) and murine TM (mTM) cells in vitro to enhance the robustness and translatability of murine glaucoma models. In this in vitro study, we compared primary hTM and mTM cells under simulated physiological and pathological conditions by exposing both cell types to the glucocorticoid dexamethasone (DEX) and Transforming Growth Factor β (TGFB2), both of which are critical in the pathogenesis of several ophthalmological diseases, including glaucoma. Phagocytic properties were assessed using microbeads. Cells were analyzed through immunocytochemistry (ICC) and Western blot (WB) to evaluate the expression of extracellular matrix (ECM) components, such as Fibronectin 1 (FN1) and Collagen IV (COL IV). Filamentous-Actin (F-Act) staining was used to analyze cross-linked actin network (CLAN) formation. Additionally, we evaluated cytoskeletal components, including Vimentin (VIM), Myocilin (MYOC), and Actin-alpha-2 (ACTA2). Our results demonstrated significant similarities between human and murine TM cells in basic morphology, phagocytic properties, and ECM and cytoskeletal component expression under both homeostatic and pathological conditions in vitro. Both human and murine TM cells exhibited epithelial-to-mesenchymal transition (EMT) after exposure to DEX or TGFB2, with comparable CLAN formation observed in both species. However, there were significant differences in FN1 and MYOC induction between human and murine TM cells. Additionally, MYOC expression in hTM cells depended on fibronectin coating. Our study suggests that murine glaucoma models are potentially translatable to human TM. The observed similarities in ECM and cytoskeletal component expression and the comparable EMT response and CLAN formation support the utility of murine models in glaucoma research. The differences in FN1 and MYOC expression between hTM and mTM warrant further investigation due to their potential impact on TM properties. Overall, this study provides valuable insights into the species-specific characteristics of TM and highlights opportunities to refine murine models for better relevance to human glaucoma.
... For all ages, the TM is known to be a major source of outflow resistance in the eye and the major source of elevated outflow resistance in ocular hypertension. 22 Segmental AHO is also associated with segmental regions of the TM showing locally increased or decreased AHO resistance. 23 Therefore, TM bypass or ablation is an effective surgery to lower IOP in adult and childhood glaucoma patients. ...
Purpose:
To compare aqueous humor outflow (AHO) pathway patterns between eyes of childhood glaucoma patients and non-glaucomatous patients receiving cataract surgery.
Methods:
Aqueous angiography was performed in childhood glaucoma eyes (n = 5) receiving glaucoma surgery and in pediatric (n = 1) and healthy adult (n = 5) eyes receiving cataract surgery. Indocyanine green (0.4%) was introduced into the anterior chamber, and AHO was imaged using an angiographic camera (SPECTRALIS HRA+OCT with Flex Module). Images were acquired and analyzed (ImageJ with Analyze Skeleton 2D/3D plugin) from the nasal sides of the eyes, the usual site of glaucoma angle procedures. Image analysis endpoints included AHO vessel length, maximum vessel length, number of branches, number of branch junctions, and vessel density.
Results:
Qualitatively, childhood glaucoma eyes demonstrated lesser AHO pathway arborization compared to pediatric and adult eyes without glaucoma. Quantitatively, childhood glaucoma and healthy adult cataract eyes showed similar AHO pathway average branch lengths and maximum branch lengths (P = 0.49-0.99). However, childhood glaucoma eyes demonstrated fewer branches (childhood glaucoma, 198.2 ± 35.3; adult cataract, 506 ± 59.5; P = 0.002), fewer branch junctions (childhood glaucoma, 74.6 ± 13.9; adult cataract, 202 ± 41.2; P = 0.019), and lower vessel densities (childhood glaucoma, 8% ± 1.4%; adult cataract, 17% ± 2.5%; P = 0.01).
Conclusions:
Childhood glaucoma patients demonstrated fewer distal AHO pathways and lesser AHO pathway arborization. These anatomical alternations may result in a new source of trabecular meshwork-independent AHO resistance in this disease cohort.
Translational relevance:
Elevated distal outflow pathway resistance due to decreased AHO pathway arborization may explain some cases of failed trabecular bypass surgery in childhood glaucoma.
... Situated at the iridocorneal angle in the anterior chamber of the eye (Fig. 1), the trabecular meshwork (TM) and Schlemm's canal (SC) inner wall endothelium form the central functional unit of the conventional outflow pathway [1,2]. The two tissues jointly regulate aqueous humor outflow resistance and thereby intraocular pressure [3], with the bulk of this resistance localized to the deepest aspect of the juxtacanalicular tissue (JCT) region of the TM and SC inner wall basal lamina [4,5] (Fig. 2). The complex structural and biochemical environment of the multi-layered TM and SC is governed by a specialized extracellular matrix (ECM). ...
This review highlights the importance of extracellular matrix (ECM) biomaterials in understanding the biology of human trabecular meshwork (TM) and Schlemm's canal (SC) cells under normal and simulated glaucoma-like conditions. We provide an overview of recent progress in the development and application of state-of-the-art 3D ECM biomaterials including cell-derived ECM, ECM scaffolds, Matrigel, and ECM hydrogels for studies of TM and SC cell (patho)biology. Such bioengineered platforms enable accurate and reliable modeling of tissue-like cell-cell and cell-ECM interactions. They bridge the gap between conventional 2D approaches and in vivo/ex vivo models, and have the potential to aid in the identification of the causal mechanism(s) for outflow dysfunction in ocular hypertensive glaucoma. We discuss each model's benefits and limitations, and close with an outlook on future directions.
... Introduction Intraocular pressure (IOP) is maintained by the balance between the level of aqueous humor produced by the ciliary body and the rate by which it exits the eye through the trabecular meshwork (TM) in the anterior portion of the eye [1][2][3][4]. The movement of aqueous humor outflow is considered to be segmental, or non-uniform, around the circumference of the eye so that regions of high flow are intermingled with regions of low and medium flow [5][6][7][8]. ...
In this study we used a spatial transcriptomics approach to identify genes specifically associated with either high or low outflow regions in the trabecular meshwork (TM) that could potentially affect aqueous humor outflow in vivo. High and low outflow regions were identified and isolated from organ cultured human anterior segments perfused with fluorescently-labeled 200 nm FluoSpheres. The NanoString GeoMx Digital Spatial Profiler (DSP) platform was then used to identified genes in the paraffin embedded tissue sections from within those regions. These transcriptome analyses revealed that 16 genes were statistically upregulated in high outflow regions and 57 genes were statistically downregulated in high outflow regions when compared to low outflow regions. Gene ontology enrichment analysis indicated that the top three biological categories of these differentially expressed genes were ECM/cell adhesion, signal transduction, and transcription. The ECM/cell adhesion genes that showed the largest differential expression (Log2FC ±1.5) were ADAM15, BGN, LDB3, and CRKL. ADAM15, which is a metalloproteinase that can bind integrins, was upregulated in high outflow regions, while the proteoglycan BGN and two genes associated with integrin signaling (LDB3, and CRKL) were downregulated. Immunolabeling studies supported the differential expression of ADAM15 and showed that it was specifically upregulated in high outflow regions along the inner wall of Schlemm’s canal and in the juxtacanalicular (JCT) region of the TM. In addition to these genes, the studies showed that genes for decorin, a small leucine-rich proteoglycan, and the α8 integrin subunit were enriched in high outflow regions. These studies identify several novel genes that could be involved in segmental outflow, thus demonstrating that digital spatial profiling could be a useful approach for understanding segmental flow through the TM. Furthermore, this study suggests that changes in the expression of genes involved in regulating the activity and/or organization of the ECM and integrins in the TM are likely to be key players in segmental outflow.
... The TM forms most of the resistance to aqueous humour outflow and thus plays an important role in the regulation of intraocular pressure, one of the leading risk factors for glaucoma. 39 TM cells function to phagocytose debris in aqueous humour as well as secrete specific enzymes and extracellular matrix. 40 Decreased cellularity and dysfunction of TM cells, along with abnormal extracellular matrix accumulation and increased TM stiffness, are observed with ageing and in primary open angle glaucoma. ...
Adult stem cells, present in various parts of the human body, are undifferentiated cells that can proliferate and differentiate to replace dying cells within tissues. Stem cells have specifically been identified in the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and orbital fat. The identification of ocular stem cells broadens the potential therapeutic strategies for untreatable eye diseases. Currently, stem cell transplantation for corneal and conjunctival diseases remains the most common stem cell‐based therapy in ocular clinical management. Lens epithelial stem cells have been applied in the treatment of paediatric cataracts. Several early‐phase clinical trials for corneal and retinal regeneration using ocular stem cells are also underway. Extensive preclinical studies using ocular stem cells have been conducted, showing encouraging outcomes. Ocular stem cells currently demonstrate great promise in potential treatments of eye diseases. In this review, we focus on the identification, characterisation, and therapeutic application of adult stem cells in the eye.
... Physiologically, with any change in outflow resistance, the outflow pathways respond by altering the outflow facility so that aqueous outflow balances the inflow rate and a steady IOP is maintained. However, with a significant increase in outflow resistance, the balance between outflow facility and aqueous inflow is achieved only at significantly high IOP levels [5,6]. ...
Introduction:
Elevated intraocular pressure (IOP) is a well-recognized risk factor for development of primary open angle glaucoma (POAG), a leading cause of irreversible blindness. Ocular hypertension is associated with excessive extracellular matrix (ECM) deposition in trabecular meshwork (TM) resulting in increased aqueous outflow resistance and elevated IOP. Hence, therapeutic options targeting ECM remodeling in TM to lower IOP in glaucomatous eyes are of considerable importance.
Areas covered:
This paper discusses the complex process of ECM regulation in TM and explores promising therapeutic targets. The role of Transforming Growth Factor-β as a central player in ECM deposition in TM is discussed. We elaborate the key regulatory processes involved in its activation, release, signaling, and cross talk with other signaling pathways including Rho GTPase, Wnt, integrin, cytokines, and renin-angiotensin-aldosterone. Further, we summarize the therapeutic agents that have been explored to target ECM dysregulation in TM.
Expert opinion:
Targeting molecular pathways to reduce ECM deposition and/or enhance its degradation are of considerable significance for IOP lowering. Challenges lie in pinpointing specific targets and designing drug delivery systems to precisely interact with pathologically active/inactive signaling. Recent advances in monoclonal antibodies, fusion molecules, and vectored nanotechnology offer potential solutions.
... Element quality assessment and mesh density analyses were all performed prior to simulations [68] . The hydraulic conductivity of 2.0 μl/min/mmHg/cm 2 [107] , 2.5 mmHg/μl/min/cm 2 [108] , and 90 0 0 ×10 −11 cm 2 sec/g [29] , which is responsible for ∼10% of total outflow resistance [ 109 , 110 ], were programmed for the TM, JCT, and SC inner wall, respectively [33] . ...
The conventional aqueous outflow pathway, encompassing the trabecular meshwork (TM), juxtacanalicular connective tissue (JCT), and inner wall endothelium of Schlemm's canal (SC), governs intraocular pressure (IOP) regulation. This study targets the biomechanics of low-flow (LF) and high-flow (HF) regions within the aqueous humor outflow pathway in normal and glaucomatous human donor eyes, using a combined experimental and computational approach. LF and HF TM/JCT/SC complex tissues from normal and glaucomatous eyes underwent uniaxial tensile testing. Dynamic motion of the TM/JCT/SC complex was recorded using customized green-light optical coherence tomography during SC pressurization in cannulated anterior segment wedges. A hyperviscoelastic model quantified TM/JCT/SC complex properties. A fluid-structure interaction model simulated tissue-aqueous humor interaction. FluoSpheres were introduced into the pathway via negative pressure in the SC, with their motion tracked using two-photon excitation microscopy. Tensile test results revealed that the elastic moduli of the LF and HF regions in glaucomatous eyes are 3.5- and 1.5-fold stiffer than the normal eyes, respectively. The FE results also showed significantly larger shear moduli in the TM, JCT, and SC of the glaucomatous eyes compared to the normal subjects. The LF regions in normal eyes demonstrated larger elastic moduli compared to the HF regions in glaucomatous eyes. The resultant strain in the outflow tissues and velocity of the aqueous humor in the FSI models were in good agreement with the digital volume correlation and 3D particle image velocimetry data, respectively. This study uncovers stiffer biomechanical responses in glaucomatous eyes, with LF regions stiffer than HF regions in both normal and glaucomatous eyes.
... While the pores in the inner wall endothelial cells of SC account for only approximately 10% of the resistance in the conventional aqueous outflow pathway [23], changes in extracellular matrix (ECM) production in the JCT or the greater SC basement membrane can impact the funneling of aqueous humor through the inner wall pores, leading to increased resistance and decreased outflow [18,24]. It has been noted that excessive ECM accumulation in the JCT and trabecular beam thickening in glaucoma patients contributes to abnormally high outflow resistance in this region in primary open-angle glaucoma (POAG) patients [25,26]. ...
Purpose
The conventional aqueous outflow pathway, which includes the trabecular meshwork (TM), juxtacanalicular tissue (JCT), and the inner wall endothelium of Schlemm's canal (SC), regulates intraocular pressure (IOP) by controlling the aqueous humor outflow resistance. Despite its importance, our understanding of the biomechanics and hydrodynamics within this region remains limited. Fluid-structure interaction (FSI) offers a way to estimate the biomechanical properties of the JCT and SC under various loading and boundary conditions, providing valuable insights that are beyond the reach of current imaging techniques.
Methods
In this study, a normal human eye was fixed at a pressure of 7 mm Hg, and two radial wedges of the TM tissues, which included the SC inner wall basement membrane and JCT, were dissected, processed, and imaged using 3D serial block-face scanning electron microscopy (SBF-SEM). Four different sets of images were used to create 3D finite element (FE) models of the JCT and inner wall endothelial cells of SC with their basement membrane. The outer JCT portion was carefully removed as the outflow resistance is not in that region, leaving only the SCE inner wall and a few μm of the tissue, which does contain the resistance. An inverse iterative FE algorithm was then utilized to calculate the unloaded geometry of the JCT/SC complex at an aqueous humor pressure of 0 mm Hg. Then in the model, the intertrabecular spaces, pores, and giant vacuole contents were replaced by aqueous humor, and FSI was employed to pressurize the JCT/SC complex from 0 to 15 mm Hg.
Results
In the JCT/SC complex, the shear stress of the aqueous humor is not evenly distributed. Areas proximal to the inner wall of SC experience larger stresses, reaching up to 10 Pa, while those closer to the JCT undergo lower stresses, approximately 4 Pa. Within this complex, giant vacuoles with or without I-pore behave differently. Those without I-pores experience a more significant strain, around 14%, compared to those with I-pores, where the strain is roughly 9%.
Conclusions
The distribution of aqueous humor wall shear stress is not uniform within the JCT/SC complex, which may contribute to our understanding of the underlying selective mechanisms in the pathway.
... Directly adjacent to the JCT is a monolayer of endothelial-like cells on top of a basement membrane that forms the inner wall of Schlemm's Canal (SC). These last two layers of the TM are considered to be the major sites involved in regulating the outflow of aqueous humor and intraocular pressure (IOP) (4) and are also the regions where profibrotic changes are thought to lead to the pathogenesis of primary open angle glaucoma (POAG) (5). ...
Primary open angle glaucoma (POAG) is a progressive and chronic disease exhibiting many of the features of fibrosis. The extracellular matrix (ECM) in the trabecular meshwork (TM) undergoes extensive remodeling and enhanced rigidity, resembling fibrotic changes. In addition, there are changes associated with myofibroblast activation and cell contractility that further drives tissue fibrosis and stiffening. This review discusses what is known about the integrins in the TM and their involvement in fibrotic processes.
Increased adhesion forces between trabecular meshwork (TM) cells and the extracellular matrix (ECM) in the human outflow pathway are associated with elevated intraocular pressure (IOP), a key risk factor for primary open-angle glaucoma (POAG). This study examines how matrix stiffness affects traction forces and collagen fibril organization in normal and glaucomatous TM cells using collagen gels with stiffness levels of 4.7 and 27.7 kPa. Normal high-flow (HF) TM/juxtacanalicular tissue (JCT) cells showed greater traction forces on the stiffer gels, whereas glaucomatous HF TM/JCT cells generated greater forces on the softer gels. These differences correlated with findings that normal cells are ∼1.6-fold stiffer than their glaucomatous counterparts. Glaucomatous cells also exhibited anisotropic collagen fibril alignment and distinct cytoskeletal dynamics. These results suggest that altered mechanosensitivity and ECM reorganization in glaucomatous TM cells may contribute to promoting ECM stiffening, elevated IOP, and disease progression, highlighting potential therapeutic strategies.
Open-angle glaucoma (OAG) is a leading cause of permanent blindness worldwide, and surgical interventions that restore the natural aqueous humor outflow pathway have emerged as promising treatment options. Therefore, we aimed to analyze the efficacy and safety profile of specific antiglaucoma surgeries, namely canaloplasty (ab interno and ab externo techniques) and gonioscopy-assisted transluminal trabeculotomy (GATT), in surgical treatment patients with primary and secondary OAG. Consequently, a systematic review of the recent literature was conducted using online databases. The effectiveness of the surgeries was assessed by reductions in intraocular pressure (IOP) measurements and decreased use of antiglaucoma eye drops preoperatively and postoperatively. The safety profile of these procedures was evaluated by recording the incidence of specific intraoperative and postoperative complications. Independent studies have shown that ab interno and ab externo canaloplasty procedures and GATT effectively lower IOP and decline medications burden. Therefore, given the favorable safety profiles, canaloplasty and GATT are associated with low incidences of postoperative adverse events and exhibit comparable safety characteristics. However, additional research, including a well-conducted randomized controlled trial comparing ab externo and ab interno canaloplasty with GATT, is required to validate our findings.
Ab interno trabeculectomy via Trabectome (MicroSurgical Technology, Redmond, WA), gonioscopy-assisted transluminal trabeculotomy (GATT), or goniotomy with Kahook Dual Blade (New World Medical, Inc., Rancho Cucamonga, CA) are minimally invasive glaucoma surgeries (MIGS) that increase the conventional outflow pathway in patients with ocular hypertension, open-angle glaucomas, and certain types of angle closure glaucoma by removing or ablating the trabecular meshwork (TM), which is considered the greatest site of resistance to aqueous outflow. Ab interno trabeculectomy with Trabectome ablates the TM and inner wall of the Schlemm’s canal (IWSC) and accesses multiple collector channels in multiple clock hours (typically nasally and/or inferiorly) using an electrode handpiece coupled with an irrigation/aspiration port, while GATT relies on a catheter or prolene suture threaded through Schlemm’s canal (SC) to incise the TM and IWSC, typically 360 degrees. KDB goniotomy is a single-use ophthalmic knife that lifts and completely excises the trabecular meshwork in multiple clock hours of treatment (typically nasally and/or inferiorly) with a similar mechanism of action as Trabectome and GATT. In all these techniques, these blebless procedures generate direct communication between the anterior chamber and multiple aqueous collector channels within Schlemm’s canal, restoring more physiologic outflow in patients where the primary site of aqueous resistance is felt to be at the level of the TM. In contrast to traditional incisional goniotomy, which uses a microvitreoretinal blade in pediatric glaucomas, histologic studies on these more recent modifications to this angle-based procedure have revealed clean edges and minimal residual leaflets of tissue (which, if left in place, can produce inflammation, fibrosis, and consequently, increased risk of scarring of the induced surgical cleft and functional failure of the procedure). Each procedural variation—which can be thought of conceptually as variations upon a theme of either ablation, incision, or excision of a section of trabecular meshwork tissue to produce an incomplete or complete window into Schlemm’s canal— can be utilized in mild, moderate, or severe open-angle glaucoma, ocular hypertension, and in some cases of angle-closure where there is no active neovascularization, and goniosynechialysis is still possible. Now 20 years since the advent of the Trabectome, high-quality Level-I prospective evidence, systematic reviews, meta-analyses, and randomized controlled trials (RCTs) by now exist regarding the safety and effectiveness of ab interno goniotomy, Trabectome, and GATT for the treatment of primary angle closure glaucoma (PACG) and primary open-angle glaucoma. Studies have also suggested that angle procedures are effective in congenital glaucoma and secondary open-angle glaucoma. Goniotomy and trabeculotomy can be combined elegantly with phacoemulsification in patients with visually significant cataract and uncontrolled intraocular pressure or patients intolerant of glaucoma medications. Surgical techniques are similar between the procedures and easily integrate with incisions and techniques used in phacoemulsification cataract surgery, with the added requirements of patient and microscope positioning for direct visualization of angle structures, surgeon comfort with identification of key landmarks, and proficiency with surgical gonioscopy. The majority of surgical complications encountered are typically transient and self-limited, as compared to the more potentially serious complication profile of the majority of glaucoma surgical alternatives. Ab interno angle procedures are favorable because of comparable efficacy, better safety profile, and shorter operating time, are typically conducted under local (intracameral) anesthesia, offer patients faster visual recovery, and lack implant- and bleb-related complications when compared to traditional incisional glaucoma surgery. The authors hope that this chapter helps non-MIGS surgeons better appreciate and approach the procedure both conceptually and practically and can help new MIGS surgeons approach the practical aspects of approaching these techniques with greater confidence, while maintaining a broader perspective of the role of angle-based procedures in the surgical management of glaucoma.
The development of new medical instruments for surgical treatment of glaucoma patients is an urgent scientific and applied task of modern mechanical engineering, since the number of visually impaired people in Ukraine due to glaucoma is growing every year. Today, the phenomenon of ultrasonic cavitation is widely used in technology, which occurs when high-intensity ultrasonic vibrations are introduced into a liquid and provides high-quality removal of contaminants from surfaces, disinfection, fine atomization, the formation of intense microcurrents, etc. Our work investigates changes in biomechanical reactions as a result of minimally invasive glaucoma surgery - expansion of physiological pathways for the outflow of intraocular fluid (trabecular apparatus, Schlemm's canal, etc.) using procedures using ultrasonic cavitation. By modernizing the phacoemulsifier, an ultrasonic glaucoma scalpel was created, which will allow cleaning the pores of the trabecular meshwork, restoring and maintaining its elasticity, reducing resistance to the outflow of ocular fluid, and reducing intraocular pressure. The use of such a tool will help ophthalmologists perform minimally invasive interventions aimed at normalizing the level of intraocular pressure in a less invasive and safer way, which will contribute to the prevention of progression and successful treatment of glaucoma. The development of the latest medical tools will make it possible to develop individual treatment strategies based on the specific needs and severity of the disease of each patient and create a perfect system of treatment for glaucoma patients.
Glaucoma is defined as a disturbance of the structural or functional integrity of the optic nerve that causes characteristic changes in the optic nerve leading to permanent defects in the visual field. If left untreated, it can lead to blindness. Glaucoma is the leading cause of irreversible blindness throughout the world and is second only to macular degeneration in the USA. Amongst the risk factors, elevated intraocular pressure, thin corneal thickness, increasing age, and a large cup-to-disc ratio are established. Concerning its pathogenesis, mechanical and vascular theories are fundamental. In addition, many other mechanisms contribute; such as immune factors, apoptosis, glutamate-induced excitotoxicity, free radicals, nitric acid synthase, and genetic mutations. To assess glaucoma; tonometry, gonioscopy, examination of optic disc, and visual field testing are essential. Animal models for glaucoma have been used to study the pathophysiological basis of glaucoma, aqueous humor dynamics, the trabecular meshwork, the ciliary muscle, and retinal ganglion cells. Animal experiments have provided a worthwhile means of evaluating diagnostic modalities and treatments, both medical and surgical.
Background/Objectives: Primary open-angle glaucoma (POAG), if caused by elevated intraocular pressure (IOP), may require a trabeculotomy (LOT), in which the trabecular meshwork (TM) and Schlemm’s canal (ISC) are incised. However, the association between the incision angle and outcomes remains unclear. Therefore, in this study, we investigated the surgical outcomes of a trabeculotomy combined with cataract surgery in patients with POAG over a 12-month follow-up period. Methods: We included 66 patients (corresponding to 83 eyes) with POAG who underwent trabeculotomy ab externo with a metal probe (M-LOT: 120° incision of the TM and ISC), ab interno with a Kahook Dual Blade® (K-LOT: 180° incision of the TM and ISC), or ab interno with a 5-0 nylon suture (S-LOT: 360° incision of the TM and ISC) between January 2015 and December 2022. We assessed IOP, the percentage reduction from preoperative IOP, the number of IOP-lowering medications taken, the incidence of postoperative complications, and the success rate using Kaplan–Meier survival analysis. Results: The median IOP was significantly lower than the baseline across all three groups. The number of IOP-lowering medications taken was significantly reduced only in the M-LOT group. The mean percentage reduction from the preoperative IOP in the K-LOT group was significantly lower than that in the M and S-LOT groups. Beween those subjected to an ab ineterno LOT, the S-LOT group demonstrated a significantly higher rate of IOP reduction 12 months after the operation compared to the K-LOT group. Kaplan–Meier cumulative survival analyses revealed a lower success rate for the K-LOT group than for the M and S-LOT groups. The M-LOT group had the lowest incidence of hyphema and IOP spikes, whereas the S-LOT group had the highest incidence of these complications. Conclusions: The M-, K-, and S-LOTs had different surgical outcomes during the 12 months of follow-up, with the M-LOT group showing the fewest complications. These results will help in selecting the most suitable trabeculotomy strategy for patients with POAG. Based on the postoperative outcomes of the ab interno K- and S-LOTs, a wider incision of the TM and ISC leads to effective IOP reduction.
Intraocular pressure (IOP) is the most important modifiable risk factor for glaucoma and fluctuates considerably within patients over short and long time periods. Our field’s understanding of IOP has evolved considerably in recent years, driven by tonometric technologies with increasing accuracy, reproducibility, and temporal resolution that have refined our knowledge regarding the relationship between IOP and glaucoma risk and pathogenesis. The goal of this article is to review the published literature pertinent to the following points: 1) the factors that determine IOP in physiologic and pathologic states; 2) technologies for measuring IOP; 3) scientific and clinical rationale for measuring diverse IOP metrics in patients with glaucoma; 4) the impact and shortcomings of current standard-of-care IOP monitoring approaches; 5) recommendations for approaches to IOP monitoring that could improve patient outcomes; and 6) research questions that must be answered to improve our understanding of how IOP contributes to disease progression. Retrospective and prospective data, including that from landmark clinical trials, document greater IOP fluctuations in glaucomatous than healthy eyes, tendencies for maximal daily IOP to occur outside of office hours, and, in addition to mean and maximal IOP, an association between IOP fluctuation and glaucoma progression that is independent of mean in-office IOP. Ambulatory IOP monitoring, measuring IOP outside of office hours and at different times of day and night, provides clinicians with discrete data that could improve patient outcomes. Eye care clinicians treating glaucoma based on isolated in-office IOP measurements may make treatment decisions without fully capturing the entire IOP profile of an individual. Data linking home blood pressure monitors and home glucose sensors to dramatically improved outcomes for patients with systemic hypertension and diabetes and will be reviewed as they pertain to the question of whether ambulatory tonometry is positioned to do the same for glaucoma management. Prospective randomized controlled studies are warranted to determine whether remote tonometry-based glaucoma management might reduce vision loss and improve patient outcomes.
Glaucoma, a progressive eye disease leading to irreversible blindness, currently affects over 70 million people globally. Elevated intraocular pressure (IOP) is implicated in its development. IOP is carefully regulated by the trabecular meshwork (TM). However, studying TM behavior has been limited to traditional tissue culture studies or costly ex vivo cultures of animal and donor eyes. Developing novel functional TM models could enhance cell/tissue behavior understanding and aid therapeutic development for glaucoma. In this study, we 3D printed a simplified and reproducible model of the human TM (hTM) and studied hTM cell behavior under static and dynamic cultures. Gelatin Methacryloyl bioinks proved suitable for printing with viable and proliferative hTM cells expressing crucial marker genes in response to glucocorticoid induction. This, to our knowledge, is the first functional 3D printed hTM model and aims to facilitate TM research. Moreover, this easily reproducible model could also be applicable in the study of numerous other cell types throughout the body.
The lack of infrastructure and accessibility in medical treatments has been considered as a global chronic issue since the concept of treatment and prevention was presented. After the COVID-19 pandemic, the medical reaction capability for epidemic outbreak/spread has been spotlighted as a critical issue to the fore worldwide. To reduce the burden on the medical system from the simultaneous disease emergence, the personalized wearable electronic systems have arisen as the next-generation biomedical monitoring/treating equipment for infectious diseases at the initial stage. In particular, electronic skin (e-skin) with its potential for multifunctional extendibility has been enabled to be applied to next-generation long-term healthcare devices with real-time biosignal sensing. Here, we introduce the recent enhancements of various e-skin systems for healthcare applications in terms of material types and device structures, including sensor components, biological signal sensing mechanisms, applicable technological advancements, and medical utilization.
Purpose: To investigate gel stent implantation with and without intraoperative sustained-release mitomycin C (MMC SR) in a rabbit model for gel stent implantation, and to examine aqueous humor outflow (AHO) postimplantation. Methods: Four groups of rabbits were included. Group 1 was untreated (control). Groups 2, 3, and 4 received the gel stent without MMC, with MMC solution (subconjunctival injection), and with MMC SR (subconjunctival injection), respectively. Intraocular pressure (IOP) and AHO were assessed via tonometry and indocyanine green-based angiography, respectively. The main efficacy measure was change in IOP from baseline. Results: Following gel stent implantation, Groups 2, 3, and 4 maintained ≥20% IOP reduction (response) for a median duration of 1 week, 6.5 weeks, and 30 weeks, respectively. Angiography showed normal aqueous humor drainage (Group 1) beginning at the perilimbal trabecular plexus and continuing posteriorly to episcleral outflow vessels. Following implantation, drainage occurred preferentially and directly into the subconjunctival bleb. Conclusions: Gel stent implantation with MMC SR was most effective in achieving sustained, long-term IOP reduction in the rabbit model, compared with implantation with or without MMC solution. Bleb presence and the postimplantation aqueous angiography results indicated redirection of the AHO to the subconjunctival vasculature and presumed lymphatics, suggesting efficient glaucoma filtration to lower IOP in this model. This rabbit model and aqueous angiography may help refine understanding of the mechanism of action of minimally invasive glaucoma surgeries and ultimately translate to improved surgical devices and procedures for patients with glaucoma.
Glaucoma continues to be the leading cause of irreversible blindness worldwide. The main proven risk factor is elevated intraocular pressure. But questions remain on the impact of various somatic diseases and their correction on the course of glaucoma. This review provides information on the metabolic syndrome and considers some of its components and their correction in patients with glaucoma.
The metabolic syndrome includes arterial hypertension, diabetes mellitus, dyslipidemia and obesity. The presented experimental and clinical studies show that the components of the metabolic syndrome correlate with glaucoma. Such components of the metabolic syndrome as arterial hypertension and diabetes mellitus are without doubt risk factors for the development of glaucoma. The opinions of researchers regarding whether obesity and dyslipidemia can also be considered risk factors are ambiguous. Recently, however, interest in them has increased due to in-depth study of the microbiome, since changes in it play a certain role in the development of neurodegenerative diseases of the central nervous system and the retina. Several studies have noted that being overweight and obese is associated with a risk of developing glaucoma. Indicators of lipid metabolism also showed a certain relationship with the risk of developing glaucoma. Treatment of lipid metabolism disorders commonly involves the use of statin drugs. Recent studies show that they may have more than just a hypolipidemic effect. A number of studies has demonstrated that the use of statins improves blood circulation and has a neuroprotective effect.
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.
The Hippo signalling pathway, an evolutionarily conserved kinase cascade, has been shown to be crucial for cell fate determination, homeostasis and tissue regeneration. Recent experimental and clinical studies have demonstrated that the Hippo signalling pathway is involved in the pathophysiology of ocular diseases. This article provides the first systematic review of studies on the regulatory and functional roles of mammalian Hippo signalling systems in eye diseases. More comprehensive studies on this pathway are required for a better understanding of the pathophysiology of eye diseases and the development of effective therapies.
The progressive degradation in the trabecular meshwork (TM) is related to age-related ocular diseases like primary open-angle glaucoma. However, the molecular basis and biological significance of the aging process in TM have not been fully elucidated. Here, we established a dynamic single-cell transcriptomic landscape of aged macaque TM, wherein we classified the outflow tissue into 12 cell subtypes and identified mitochondrial dysfunction as a prominent feature of TM aging. Furthermore, we divided TM cells into 13 clusters and performed an in- depth analysis on cluster 0, which had the highest aging score and the most significant changes in cell proportions between the two groups. Ultimately, we found that the APOE gene was an important differentially expressed gene in cluster 0 during the aging process, highlighting the close relationship between cell migration and extracellular matrix regulation, and TM function. Our work further demonstrated that silencing the APOE gene could increase migration and reduce apoptosis by releasing the inhibition on the PI3K-AKT pathway and downregulating the expression of extracellular matrix components, thereby increasing the aqueous outflow rate and maintaining intraocular pressure within the normal range. Our work provides valuable insights for future clinical diagnosis and treatment of glaucoma.
Purpose
This study aimed to histologically compare the status of Schlemm's canal (SC) and Schlemm's canal endothelial (SCE) cells between trabeculectomy specimens from patients with primary open-angle glaucoma (POAG) and exfoliation glaucoma (EXG).
Methods
A total of 182 eyes from 152 patients with POAG and 138 eyes from 116 patients with EXG underwent immunohistochemical staining for thrombomodulin. Equal numbers of cases were selected from both groups using propensity score matching. The following parameters were evaluated: total SC length, staining positive and negative SC length (PSC and NSC, respectively), opened and closed SC length, staining positive and opened SC length, staining positive and closed SC length, staining negative and opened SC length (NOSC), and staining negative and closed SC length.
Results
After matching for age and gender, 87 cases were selected in each group. The EXG group had significantly higher preoperative IOP and medication scores. PSC was significantly longer in the POAG group, while NSC and NOSC were longer in the EXG group. Multiple regression analysis of these 174 cases revealed that PSC was significantly shorter in the EXG group. After matching for age, gender, preoperative IOP, and medication score, 64 cases were selected in each group, and NOSC was significantly longer in the EXG group.
Conclusions
These findings suggest that in EXG, SCE loss occurs independently of background factors such as aging and medication use. The loss of SCE may have a more critical impact on IOP elevation in EXG compared to POAG.
Oxidative stress occurs through an imbalance between the generation of reactive oxygen species (ROS) and the antioxidant defense mechanisms of cells. The eye is particularly exposed to oxidative stress because of its permanent exposure to light and due to several structures having high metabolic activities. The anterior part of the eye is highly exposed to ultraviolet (UV) radiation and possesses a complex antioxidant defense system to protect the retina from UV radiation. The posterior part of the eye exhibits high metabolic rates and oxygen consumption leading subsequently to a high production rate of ROS. Furthermore, inflammation, aging, genetic factors, and environmental pollution, are all elements promoting ROS generation and impairing antioxidant defense mechanisms and thereby representing risk factors leading to oxidative stress. An abnormal redox status was shown to be involved in the pathophysiology of various ocular diseases in the anterior and posterior segment of the eye. In this review, we aim to summarize the mechanisms of oxidative stress in ocular diseases to provide an updated understanding on the pathogenesis of common diseases affecting the ocular surface, the lens, the retina, and the optic nerve. Moreover, we discuss potential therapeutic approaches aimed at reducing oxidative stress in this context.
We review the natural pathways and glaucoma implant-based artificial pathways of aqueous humour in the eye from a microfluidic perspective.
Glaucoma, marked by its intricate association with intraocular pressure (IOP), stands as a predominant cause of non-reversible vision loss. In this review, the physiological relevance of IOP is detailed, alongside its potential pathological consequences. The review further delves into innovative engineering solutions for IOP monitoring, highlighting the latest advancements in wearable and implantable sensors and their potential in enhancing glaucoma management. These technological innovations are interwoven with clinical practice, underscoring their real-world applications, patient-centered strategies, and the prospects for future development in IOP control. By synthesizing theoretical concepts, technological innovations, and practical clinical insights, this review contributes a cohesive and comprehensive perspective on the IOP biosensor's role in glaucoma, serving as a reference for ophthalmological researchers, clinicians, and professionals.
Introduction:
Minimally invasive glaucoma surgeries (MIGS) are now a consolidated reality in many surgical units. The Hydrus Microstent is one of several MIGS devices bypassing trabecular outflow and had excellent results over the years. This article aims to review the key features of the Hydrus Microstent in terms of design, efficacy, and safety.
Areas covered:
The present review analyses the main characteristics of the device by evaluating the technical and physical details of its functioning. The evidence that supports a clinical decision summarizes the most influential clinical trials and the most accurate systematic reviews.
Expert opinion:
The Hydrus device has been extensively studied regarding biocompatibility and outflow potential. The subsequent clinical studies have been well-built and proved that the device effectively reduces intraocular pressure (IOP) and the eyedrop load. The device covers almost a quarter of Schlemm's canal circumference, offering at least two advantages: cannulating the Schlemm's canal provides evidence that the device has been implanted correctly; covering a larger area potentially allows to target multiple collector channels or at least areas of active outflow. This scaffold may prove more effective in naïve patients or subjects who used antiglaucoma eyedrops for a limited period, as the prolonged use of hypotonic medications has been associated with the surgical failure of ab interno microhook trabeculotomy.
PURPOSE. In a prior study, it has been reported that glaucomatous eyes have a significantly lower density of pores in the inner wall of Schlemm's canal than do normal eyes. However, in that study the glaucomatous eyes were fixed at much lower flow rates than the normal eyes, and that is now known to affect inner wall pore density. The objective of the current study was to compare the inner wall's pore density in glaucomatous and normal eyes, accounting for the effects of fixation conditions. METHODS. Outflow facility was measured in enucleated glaucomatous human eyes. Eyes were fixed under constant flow conditions, microdissected to expose the inner wall of Schlemm's canal, and prepared for scanning electron microscopy (SEM). The density and diameter of the two subpopulations of pores in the inner wall, intracellular and intercellular (or "border") pores, were measured. Data were compared with those in previous studies of normal eyes. RESULTS. As previously reported, pore density decreased with increasing postmortem time and increased with increasing volume of fixative passed through the outflow pathway and with increasing fixation time. Linear regression analysis indicated that glaucomatous eyes had less than one fifth the number of pores than normal eyes have, after accounting for the influence of volume of fixative perfused through the eyes (835 pores/mm(2) in normal eyes versus 160 pores/mm(2) in glaucomatous eyes). A nonlinear regression of pore density versus fixative volume produced a pore density at zero fixative volume that was not statistically different from zero. If true, this implies that all (or nearly all) inner wall pores observed by SEM are fixation artifacts. The density of intracellular pores and the diameter of these pores correlated with the density and diameter of the border pores, respectively. CONCLUSIONS. Inner wall pores are reduced in glaucomatous eyes. If pores are physiological structures, the elevated intraocular pressure characteristic of glaucoma may be explained by decreased porosity of the inner wall endothelium. Both border and intracellular pores seem to be induced in a similar fashion by fixation. The unlikely possibility that all inner wall pores are fixation-induced cannot be excluded. If so, a fundamental reassessment of the mechanism by which aqueous humor crosses the inner wall endothelium is necessary.
Monkeys eyes were fixed with glutaraldehyde in vivo at positive intraocular pressure of 35 or 25 mm Hg and compared with eyes fixed without a positive pressure gradient, with the use of light microscopy and transmission and scanning electron microscopy. The entire endothelial lining of the inner wall of Schlemm's canal ballooned or distended toward the external wall of the canal at positive intraocular pressure. Characteristic nuclear shapes were identified and appeared to result from the increased pressure forcing the lining away from the meshwork opposed by a restraining or anchoring effect of cytoplasmic processes attached to the subendothelial cells and trabecular meshwork. Without positive intraocular pressure endothelial cell nuclei were rounded, with many folds and notches in the nuclear membrane and were not deformed by their cytoplasmic processes. These findings suggest that the cells may be capable of elastic recoil or contraction.
We have previously established that approximately 30% of the endothelial junctions in the pericytic venules of the mouse diaphragm are open to a gap of approximately 30--60 A, and are fully permeated by hemeundecapeptide (H11P) (mol diam approximately 20 A). To estimate the size limit for molecules that can permeate these junctions, we have administered graded tracers intravenously and studied their behavior at the level of pericytic venules in bipolar microvascular fields (BMFs) in the mouse diaphragm. Horseradish peroxidase (HRP) (mol diam approximately 50 A) permeated only approximately 50% of the open junctions of the venular endothelium. Outflow through venular junctions appeared to be modest since the tracer remained restricted to the perivenular spaces. Hemoglobin (Hb, mol diam 64 x 55 x 50 A) permeated only a few (less than 5%), and ferritin (mol diam 110 A), practically none, of the endothelial junctions of the pericytic venules. The findings suggest that under normal conditions the size limit for permeant molecules for open venular junctions is approximately 60 A. Replicas of freeze-fracture preparations from appropriate regions in BMF showed that the intercellular junctions of the venular endothelium have the same organization as previously described for the corresponding segments of the microvasculature in the omentum and mesentery: discontinuous creases or grooves either free of or marked by few intramembrane particles only. Administration of histamine (topically or systemically) and 5-hydroxytryptamine (5-HT) (topically) resulted in typical focal separations of the endothelial junctions and intramural deposits of large tracer particles (carbon black) in the postcapillary venules.
PURPOSE. It has been reported that fixation conditions significantly influence the apparent pore density in the inner-wall endothelium of Schlemm's canal. In the present study, the manner in which fixation conditions affect the two subtypes of inner-wall pores, intracellular pores and intercellular (or border) pores, was investigated. METHODS. Outflow facility was measured in enucleated human eves. Eyes were fixed under "constant flow" Or constant pressure conditions, microdissected to expose the inner wall of Schlemm's canal, and prepared fur scanning electron microscopy. The density and diameter of the two subtypes of pores in the inner wall were measured. RESULTS. Intracellular pore density decreased with increasing postmortem time (P < 0.001) and increased with increasing volume of fixative passed through the outflow pathway (P < 0.001), whereas border pore density showed no dependence on these parameters (P > 0.25 and P > 0.15, respectively). Border pore density increased with increasing fixation pressure (P < 0.005), even though intracellular pore density showed no such dependence (P > 0.4). No correlation was found between outflow facility and the predictions of Poiseuille's law, Sampson's law, or the funneling theory for the hydraulic conductivity of the intracellular pores (P > 0.35) or the border pores (P > 0.1). CONCLUSIONS. The intracellular and border pores form two morphologically and functionally distinct populations in the inner wall of Schlemm's canal. The dependence of intracellular pore density on postmortem time and on volume of fixative passed through the outflow pathway suggests that these pores are artifacts of tissue fixation or processing conditions. That border pores do not depend on such conditions and that their presence is correlative with perfusion pressure suggests that this population may be nonartifactual. New histologic techniques for examining the inner wall of Schlemm's canal are necessary to determine the in vivo state of inner-wall pores and how they influence outflow facility.
Morphological and physiological studies indicate multiple routes for transport across capillary endothelium. However, the identification of the morphological counterparts of specific transport processes (or the assignment of specific transport roles to morphologically identifiable pathways) has been only partly achieved: the contribution of endothelial cell membranes to transport of water and small, lipid-insoluble molecules needs to be evaluated. The identification of the "small pore" pathway for water and lipid-insoluble molecules with the intercellular junctions still remains questionable. The contributions to total macromolecular transport of junctions, single vesicles (pinocytosis, cytopempsis), chains of vesicles, and fenestrae are not yet known.
We have previously established that approximately 30% of the endothelial junctions in the pericytic venules of the mouse diaphragm are open to a gap of approximately 30--60 A, and are fully permeated by hemeundecapeptide (H11P) (mol diam approximately 20 A). To estimate the size limit for molecules that can permeate these junctions, we have administered graded tracers intravenously and studied their behavior at the level of pericytic venules in bipolar microvascular fields (BMFs) in the mouse diaphragm. Horseradish peroxidase (HRP) (mol diam approximately 50 A) permeated only approximately 50% of the open junctions of the venular endothelium. Outflow through venular junctions appeared to be modest since the tracer remained restricted to the perivenular spaces. Hemoglobin (Hb, mol diam 64 x 55 x 50 A) permeated only a few (less than 5%), and ferritin (mol diam 110 A), practically none, of the endothelial junctions of the pericytic venules. The findings suggest that under normal conditions the size limit for permeant molecules for open venular junctions is approximately 60 A. Replicas of freeze-fracture preparations from appropriate regions in BMF showed that the intercellular junctions of the venular endothelium have the same organization as previously described for the corresponding segments of the microvasculature in the omentum and mesentery: discontinuous creases or grooves either free of or marked by few intramembrane particles only. Administration of histamine (topically or systemically) and 5-hydroxytryptamine (5-HT) (topically) resulted in typical focal separations of the endothelial junctions and intramural deposits of large tracer particles (carbon black) in the postcapillary venules.
Many aspects of the anatomy and histology of the normal trabecular meshwork are comparatively well known from light microscopy. Unsolved problems exist, however, such as the exact structure of the outermost layer of the meshwork and the existence of extracellular material in the intertrabecular spaces. This preliminary report considers these two problems, and indicates the potentialities of electron microscopy for assessing the detailed structure and topography of the trabecular meshwork.
Material and Methods
The material used in this investigation consists of three human and four monkey eyes. The human eyes were enucleated because of malignant melanoma of the posterior pole. In no cases were there any clinical signs of impaired outflow. All human material was fixed within 1-2 minutes after enucleation, and the monkey eyes were fixed in vivo before enucleation. The fixation procedure was the same, which is routinely used for electron microscopy. Sectioning was performed on a Porter-Blum
Salzmann,1 in 1912, published his classic text on the anatomy and histology of the eyeball, summarizing existing knowledge and describing the results of his own investigations. Little has been added to his description of the trabecular meshwork until recent years.
The trabecular meshwork has been considered to be composed of a uveal and a corneoscleral portion. The uveal meshwork borders the anterior chamber and its netlike fibers extend in an arc from the surface of the corneoscleral meshwork to the root of the iris. The meshwork is loose with large and irregular openings; the fibers appear rounded in cross section. The corneoscleral meshwork comprises the major portion of the trabeculae. The trabecular meshwork roughly is ring-shaped and in cross section is somewhat triangular, the apex being at the end of Descemet's membrane and the base of the triangle extending along a line between the scleral spur and the sclera
Using histochemical staining techniques and electron microscopy, the authors have examined the histochemical properties and ultrastructure of Bruch's membrane in 30 human eyes with an age range of 1 to 95 years. The results analyzed in three age groups (0–30 years, 31–60 years, and older than 60 years) show that there is a progressive accumulation of lipids in Bruch's membrane with relation to age. Differences were found in the specific types of lipids in individual eyes. Five eyes stained for neutral lipids alone, four stained predominantly for phospholipids, and nine stained intensely for both neutral lipids and phospholipids. The deposits were associated with the progressive destruction of the native architecture of Bruch's membrane but no correlation was identified between specific inclusions in Bruch's membrane with a particular lipid. These results are significant to age-related macular disease (ARMD), and the lipid rich barrier in Bruch's membrane is implicated as a cause of photoreceptor dysfunction and pigment epithelial detachment.
• We tested the hypothesis that obstruction of the juxtacanalicular tissues, by melanin granules in pigmentary glaucoma and by other impermeable material in primary open angle glaucoma, leads to the development of a chronic glaucomatous condition. The distribution and concentration of melanin and other impermeable materials in the juxtacanalicular tissues and elsewhere in the trabecular meshwork was determined in 13 specimens. Six specimens were from patients with pigmentary glaucoma, two from patients with pigment dispersion syndrome, and three from patients with primary open angle glaucoma, as well as two from normal subjects. The effect of these materials on flow resistance was estimated using two hydrodynamic models. In model A, the electron-lucent spaces of the juxtacanalicular tissue were assumed to be open spaces, while in model B, these spaces and spaces filled with ground substance were assumed to be gel filled. In pigmentary glaucoma, 3.5% of the pigment was found in the juxtacanalicular tissue, while 96.5% was found in the corneoscleral and uveoscleral tissues. Permeabilities calculated according to model A were much higher than those expected from estimates of outflow facility in all groups, in agreement with the previous report of Ethier et al. The gel-filled spaces available for fluid flow, as determined by model B, showed no statistically demonstrable differences (pigmentary glaucoma, 32.9%; primary open angle glaucoma, 36.6%; pigment dispersion syndrome, 43.4%; normal, 44.1%). Furthermore, the amount of pigment present in the juxtacanalicular tissue was determined to have a negligible influence on permeability. Thus, the development of the chronic glaucomatous condition cannot be directly attributed to pigment accumulation in the juxtacanalicular tissue in pigmentary glaucoma.
Schlemm's canal and the trabecular meshwork of eight human and eleven monkey eyes have been analyzed by means of electron microscopy.1.The size, shape and delineation of the lumen of Schlemm's canal is given.2.The wall of Schlemm's canal in a strict sense is a single endothelial layer. Through some of the cells there is a small channel, which connects the intertrabecular spaces with Schlemm's canal. The details of the endothelium is discussed.3.The wall in a broader sense is a 4–8 µ thick layer of endothelial cells, collagen material, homogeneous material and “empty” spaces.4.The first trabecular sheet has a very intimate relationship to the wall.5.The topography of the trabecular sheets is discussed. Data about the size of the sheets are given. The increasing thickness with age is especially stressed. The composition of the individual sheet has been evaluated and change in composition with age is noted.6.The topography, size and interconnections of the intertrabecular spaces are discussed. There exist open communications through the trabecular meshwork which connect the anterior chamber with Schlemm's canal.7.Nerves are present in different parts of the trabecular meshwork, even in immediate vicinity of the inner wall of Schlemm's canal. There are suggestions that some of the nerves are adrenergic.8.Besides red and white blood cells there are found plasma cells and mast cells in the trabecular meshwork.9.The difference in appearance of the trabecular meshwork between human and monkey eyes is noted.10.To a limited extent the structure of the trabecular meshwork is discussed with regard to the function.
Water permeability of Descemet's membrane can be calculated from the time sequence of corneal thickening. Permeabilities are calculated from data obtained in three different ways: (1) when an enucleated eye is cooled to 4°C; (2) when an in vivo eye is scraped free of endothelium and Descemet's membrane; and (3) when an excised cornea is allowed to thicken under controlled swelling pressure conditions. The calculation is based on the theory of water imbibition by swelling membranes. The calculated permeability of Descemet's membrane is reasonable in view of the structure of that membrane.
In preparations of human autopsy eyes in which Schlemm's canal was injected with plastoid or India ink-gelatine solution, the morphology of the canal and its outflow channels were newly investigated. In addition, serial sections were analyzed. In injection specimens the sagittal diameter of the canal measured on the average 370 μ. The plexus-like form was seen relatively rarely. Two types of outflow channels were distinguished: direct and indirect channels. The direct outflow channels show wider lumina at the point of origin in Schlemm's canal (70 μ on the average). They are not numerous, averaging only 4–6 in each eye. Running in an arch-like or spiral course, they do not anastomose very much with the intrascleral plexus but are connected more or less exclusively to the episcleral network. The indirect outflow channels are smaller (about 50 μ) and more numerous (15–20 in each eye ball). Originating at the canal they enter rapidly into the intrascleral capillary network. There are a few intermediate types of outflow channels (4–6). The distances between the outer channels of Schlemm's canal are 1.2 mm on the average. We believe that the indirect and direct channels also have different functions for the aqueous outflow. At the point of origin of the outflow channels the outer wall of Schlemm's canal shows torusor lip-like thickenings and sometimes short oblique septa, which are connected with the inner wall. They presumably keep the lumina open and direct the aqueous fluid into the outer channels. There are capillary anastomoses between the ciliary and intrascleral plexus, a fact which has not always been confirmed in the literature.
Methods were developed to measure albumin permeability and electrical resistance of bovine aortic endothelial cell (BAEC) monolayers cultured on porous polycarbonate filters. Permeability to 1% bovine serum albumin (Pe) was quantified by measuring the flux of fluorescent-labeled albumin with an apparatus in which there were no transmural oncotic or hydrostatic pressure gradients. The effect of passage of BAEC monolayers in culture on permeability was studied using 60 BAEC monolayers of Passage 6 to 10. There was no significant difference in Pe between passages, and the mean Pe of all monolayers was 4.5 0.5 (SEM) 10–6 cm/s. Using these same BAEC monolayers, a fluorescent technique was developed to examine en face permeability patterns. Most BAEC monolayers demonstrated diffuse permeability across the monolayer, whereas others had focal regions of enhanced permeability despite similar Pe values. In those monolayers with punctate permeability, there were 5.4 0.6 (SEM) focal regions of enhanced permeability per 1000 cells. To study the effect of culture time on monolayer integrity, electrical conductivities of nine BAEC monolayers were measured daily using a Millipore electrical resistance system. Electrical resistance increased from 4.5 ohmcm2 at Day 2 to a peak level of 11.4 ohmcm2 at Day 7 and then decreased daily to 4.0 ohmcm2 by Day 12. The in vitro BAEC monolayer has many of the transport characteristics of intact vessels, making these techniques useful in physiologic studies of the endothelial transport barrier. These methods provide relatively simple means of assessing the integrity of endothelial cell monolayers grown on porous substrates.
The resistance of the corneal stroma to the flow of water has been studied both along and across the tissue as well as at various degrees of hydration. In the investigations of flow along the stroma a marked difference in thickness was generated by allowing a part of the corneal strip to swell and the adjacent part to dry. Water equilibrium along the strip was established by the water movement from the swollen to the dry part and the profile was evened out. By repeated measurements of thickness along the strip, it was possible to estimate the flow of water. Since the relationship between thickness and swelling pressure was known, the pressure gradient along the cornea could be determined. For these determinations a new instrument was designed and is described in detail. The results showed a flow of water of approximately 0.6 × 10−3 mm/hr for a pressure gradient of 1 mm Hg/mm.The flow of water across the corneal stroma was determined by pressing 0.9% NaCl or water through the tissue while tightly sealed in a holder between two porous stainless-steel filters and collecting the water in a capillary.The resistance to flow in the two directions was found to be similar at normal hydration, but for dried cornea the resistance was higher across the tissue than along it. Since calculations show that the collagen fibrils alone offer only 1/50 of the measured resistance, it is suggested that the interfibrillar substance is responsible for the high resistance to the flow of water in the corneal stroma.
To determine the filtration characteristics of the aqueous outflow system, microspheres (0·18 μm–1·1 μm) were perfused through enucleated human and bovine eyes. The microspheres were smaller than morphologically determined flow dimensions, and yet a significant fraction of all sizes of microspheres were captured. The bovine (calf) aqueous outflow system was found to be a far more efficient filter than was the human outflow system.Combining the experimental results with morphological observations and theoretical calculations leads to the conclusion that ‘sticky wall’ interactions are responsible for much of the microsphere capture, and that the site of filtration may be distinct from the site of flow resistance. Consequently, the dimension of the sites generating flow resistance cannot be determined from filtration studies.
Albumin labelled with Evans blue and 131I was injected into the suprachoroid of rabbits. The eye, the periocular tissues and the lymph paths draining the head were investigated at different times after the injection. After 60 min, more than 60% of the labelled material had left the eye. A considerable part of the albumin entered the conjunctiva and left this by way of the conjunctival lymphatics, draining into regional lymph nodes which, in turn, drained essentially into the superficial cervical lymph vessels. Some albumin may have entered the conjunctival or the orbital blood capillaries. Movement of labelled albumin into the intraocular blood vessels played a small role, if any, in the removal of the albumin from the suprachoroid, and the drainage of labelled albumin with the aqueous humour was negligible. It is suggested that, even under normal conditions, albumin may move out of the eye through the transscleral paths demonstrated, and that these paths may permit diffusion into the eye of substances administered outside the eye. No definite conclusions regarding the net movement of albumin through the sclera can be drawn.
Elevated intraocular pressure in those with glaucoma appears to be a function of increased resistance to movement of aqueous humor through the conventional outflow pathway. The majority of resistance in both normal and glaucomatous eyes is generated in the region between the juxtacanalicular trabecular meshwork and the inner wall of Schlemm's canal. To accommodate transient elevations in pressure, we hypothesize that conventional outflow increases rapidly due to changes in complexity of intercellular junctions between cells of the inner wall of Schlemm's canal.
To test this hypothesis we examined specifically the effects of hydrostatic pressure gradients and the calcium chelator, Na2EDTA, on permeability of cultured human Schlemm's canal cell monolayers in isolation. Human Schlemm's Canal cells were isolated, cultured and then seeded onto permeable supports and maintained in culture to allow intercellular junctions to mature. With a minimum net transendothelial electrical resistance of 10 Ohm cm2, cells were placed into an Ussing-type chamber and hydraulic conductivity was calculated from pressure and flow measurements that were continuously recorded. Simultaneously, transendothelial electrical resistance was measured manually at fixed intervals. In parallel experiments, cell margins were monitored in real time by videomicroscopy.
During the baseline measurement period when cells were exposed to pressure but not Na2EDTA, hydraulic conductivity was constant but transendothelial electrical resistance decreased continuously at rate of 0.24 Ohm cm2/minute. After Na2EDTA treatment, no significant change in transendothelial electrical resistance was measured while, hydraulic conductivity of Schlemm's Canal monolayers increased significantly by 125%; corresponding to noticeable intercellular separations. Restoration of cell-cell contact was observed by videomicroscopy 30 minutes following washout of Na2EDTA and functionally after 2 hours.
Responses of Schlemm's Canal cells to pressure and calcium chelators in vitro are consistent with a role for calcium sensitive junctions in outflow resistance in vivo.
The hydrodynamic and osmotic permeabilities (for glucose) have been studied for the anterior bovine lens capsule. The filtration coefficient, Lp, was found to be 2·34±1·04×10−11 cc/sec/cm2/dyne for calf, and 1·15±0·34×10−11 for steer. On the other hand, the reflection coefficient, σ, for glucose was 0·0030±0·0019 for calf and 0·020±0·017 for steer. The effect of aging upon these respective coefficients suggests different pathways for water and glucose. No difference was observed in the σ value for d- and l-glucose. The thickness of the anterior capsule was found to be: 0·0048±0·0018 cm for calf as compared to 0·0085±0·0015 cm for steer. The filtration coefficient increases with the reciprocal of the thickness, but not in a linear fashion. This may be interpreted to mean that the anterior capsule does not grow by the accretion of homogenous and/or parallel layers. In general, the permeabilities of the reference substance to the capsule is comparable to many synthetic and biological membranes.
An apparatus is described for the rapid determination of both hydrodynamic and osmotic permeabilities of biological membranes.
A quantitative investigation, using transmission electron microscopy, was conducted to determine the relative incidence of nuclei, giant vacuoles and their pores, non-vacuolar transcellular channels and minipores in the endothelium lining the trabecular aspect of Schlemm's canal at various levels of intraocular pressure in the range 0--50 mmHg. The eyes of twelve rhesus monkeys were used and prior to intracameral fixation, the intraocular pressure was maintained at the appropriate level for one hour. The results obtained from a single section analysis showed that the incidence of giant vacuoles, vacuolar and non-vacuolar transcellular channels and minipores could be related to pressure. An analysis of serial reconstructions of samples of the vacuoles indicated that vacuolar transcellular channels became more numerous and more enlarged as pressure was increased. We concluded that the transcellular pore system serves as a major outflow route for aqueous humour.
As intraocular pressure is increased, facility of aqueous outflow decreases. For many years this fact was obscured by the often used procedure of experimental perfusion into the anterior chamber. Anterior chamber perfusion deepens the anterior chamber; retrodisplacement of the iris-lens diaphragm tenses the trabecular mesh with resulting facility increase and obscuration of the facility-reducing effect of pressure. The probable mechanism of facility reduction under experimental conditions is apposition of the inner wall of Schlemm's canal to the outer wall. Since facility of aqueous outflow is not constant at different intraocular pressures, application of poiseuille's law to the eye must be restricted to each pressure investigated.
This article reviews recent findings and current views concerning the structural aspects of microvascular permeability. The vascular endothelium is considered as a simple squamous epithelium which has acquired a remarkably high permeability to water and water soluble solutes (including macromolecules) through a characteristic process of differentiation of its cells. In terms of cellular structures, this differentiation involves an unusually large population of plasmalemmal vesicles. The evidence so far obtained indicates that these vesicles function as (1) mass-carriers of fluid and solutes across the endothelium and as (2) generators of transendothelial channels by concomitant fusion (followed by fission) with both domains (luminal and tissular) of the plasmalemma. The endothelial fenestrae of visceral capillaries are initially transendothelial channels subsequently collapsed to minimal length. The intercellular junctions of the endothelium are not detectably permeable to tracers of diam. greater than or equal to 18--20 A in capillaries, but are focally open to probes of 50--60 A diam. in postcapillary (pericytic) venules. A correlation is attempted between transendothelial channels (and fenestrae) and the pore systems postulated by the pore theory of capillary permeability. The channels appear to function as either small or large pores depending on the porosity of their associated diaphragms and on the size of local strictures along their pathway. Two main components are recognized in the analysis of capillary permeability: 1) a basic component comparable to that of other simple epithelia and involving transport across the plasmalemma and probably along the intercellular junctions (for molecules of diam. greater than or equal to 10 A); and a differentiated component which involves plasmalemmal vesicles and their derivatives (transendothelial channels and fenestrae). The postulated pores of the capillary endothelium are part of this differentiated component. The special situation found in postcapillary venules (focally open junctions) seems to be related to the role played by these vessels in inflammatory reactions.
Pressure-driven fluid flow across the arterial wall was measured to determine wall hydraulic conductivity (Lp) before and after removal of the endothelium. The thoracic aortas of rabbits, anesthetized with Nembutal, were cannulated, perfused with oxygenated Ringer solution, and removed. With one cannula connected to a capillary manometer and the other closed, the manometer meniscus shift could be used as an indication of fluid loss through the wall plus vessel volume increase (creep). The latter effect, when measured, accounted for about one-fourth of the total volume displacement. The Lp given in cm/(s.cmH2O) +/- SD, was 3.30 +/- 0.96 x 10(-8). Another method employed continuous weighing of a closed aortic segment to obtain fluid loss, and yielded an Lp of 4.07 +/- 1.3 x 10(-8), and after mechanically removing the endothelium, the Lp became 7.73 +/- 2.8 x 10(-8). Using the above data, an Lp could be calculated for aortic endothelium of 8.6 x 10(-8). This suggests that about half the total transmural pressure drop occurs across the endothelium. Scanning electronmicrographs were used to check the condition of the endothelium.
A model of Schlemm's canal was designed and analyzed mathematically. The effect of canal width on intraocular pressure was examined, and the pressure and flow distribution in the canal were calculated.
1. A quantitative basis for the currently accepted theory on the regulation of corneal hydration was derived using the technique of finite element analysis to integrate a set of coupled flow equations. The model was based on non-equilibrium thermodynamics and incorporated the transport and permeability properties of the corneal epithelium and endothelium as well as the gel properties of the central connective tissue layer. 2. Considerable errors were introduced in the prediction of corneal hydration dynamics (unsteady-state behaviour) unless allowance was made for the development of trans-stromal gradients in pressure and solute concentration. 3. Thickness of in vitro rabbit corneal epithelium and stroma were measured with an automatic specular microscope during responses to changes in the osmolarity of the tear-side bathing medium. The time course of these experiments was fitted with the mathematical model to obtain a set of membrane phenomenological coefficients and transport rates. 4. The model with the redetermined membrane parameters was tested by predicting the influence of other variations in boundary conditions with excellent match to several well-documented experimental observations, including an explanation for the slight stromal swelling observed in hibernating mammals. 5. The regulation of corneal stromal hydration can be explained accurately by balance between the dissipative flows across the serial array of corneal layers and the active HCO3 transport by the endothelium, supporting the earlier 'pump-leak' hypothesis. 6. It was found that stromal retardation of fluid flow, as well as gradients in solute concentration, significantly influences the dynamics of corneal stroma hydration. Tissue gel properties may be a more important factor in coupled transport across cell layers than generally appreciated.
Argon laser treatment was applied 360° to the trabecular meshwork of 56 patients with open-angle glaucoma, using 100 burns at a setting of 1,000 to 1,500 mW, 0.10 seconds, and 50 μ beam-diameter. 40 of 41 phakic eyes were controlled 3 months postlaser, but 27 eyes (65%) required some medical therapy. Late failure was not a problem, with the same mean pressure drop of 10 mm Hg noted at 3, 6, 12, and 18 months postlaser. No angle scarring or serious complications occurred. One phakic eye required additional surgery. 15 aphakic eyes showed less response, with 8 of 9 eyes on medical therapy 1 year after laser treatment, and the other 6 requiring further surgery. Increased tension in the trabecular meshwork is the postulated mechanism. The argon laser appears to be an effective alternative to filtration surgery in phakic eyes.
The drainage pathway of aqueous hmnour of the eye and cerebrospinal fluid (CSF) of the brain has been investigated with and without experimental procedures; and the appropriate tissues have been analysed by light-, scanning- and transmission-electron microscopy to unravel the structural basis for the resistance to, and the precise mechanism of, the outflow of these fluids. The close similarities in the origin, circulation, dynamics and drainage of the humours of the eye and brain are emphasized. In both cases, controversy has centred upon whether or not there are pores in the mesothelial lining of the canal of Schlemm and arachnoid villi which mediate the final outflow of aqueous humour and CSF respectively.Morphometrically, as revealed by scanning electron microscopy, the mesothelial lining of Schlemm's canal and the arachnoid villi are closely comparable, both being constituted by spindle-shaped cells measuring some 40–120 µm in length and 4µ12µm in width in the central region corresponding to one or more apical bulges. Transmission electron microscopy showed that in both cases the mesothelial lining forms a continuous single-layered membrane with adjacent cells joined by junctional complexes offering a restraint to the passage of experimentally introduced colloidal molecules of 10 nm or smaller size. The cells contain an apically bulging nucleus and a moderate amount of the usual intracelhdar organelles including a system of well developed cytoplasmic microfilaments presumably associated with a contractile function.The special feature of many lining cells of both Schlemm's canal and arachnoid villi is the presence of giant vacuolar structures of several micrometers in size. Arguments are advanced that these unit membrane-bound configurations are a real morphological entity and not a product of postmortem or fixation artefact. Morphogenetically, they are considered as developing from surface infoldings or invaginations on the basal aspect of the lining cells, which, by gradually enlarging, eventually open on to the apical cell surface thus cleaving the cells to form wide vacuolar transcellular channels of heterogenous dimensions with basal openings up to 4µm and apical openings up to 2·5µm in size. Our correlative physiological and morphological studies using a variety of tracer substances have shown that the bulk outflow of aqueous humour and of CSF takes place down a pressure gradient via the vacuolar transcellular channels which constitute a dynamic system of pores and act as one-way valves allowing fluid to pass from basal to apical aspects far more easily than in the reverse direction. According to this hypothesis the process of vacuolation is cyclical, and it would now seem certain that this delicate and probably pressure-sensitive vacuolation cycle, in providing the requisite number of pores at any given time, is a controlling factor in the bulk outflow of aqueous humour and CSF, and consequently in the maintenance of normal fluid pressure in the eye and brain. The role of various factors, especially hydrostatic pressure gradient, in influencing the vacuolation cycle is discussed.In contrast to the open passage provided by the unique biological system of vacuolar transcellular channels, other processes, e.g. micropinocytosis, passage through intercellular clefts or small diaphragmed fenestrations, phagocytosis, etc., would normally appear to make a relatively minor contribution in the bulk clearance of the humours of the eye and brain.Our comparative morphological and physiological studies of the eyes of a wide variety of sub-primate vertebrates have further resolved the age-old controversy between morphologists and physiologists, the former concluding a fimdamental difference between primates and lower mammals and the latter contending the essential similarity. Despite gross morphological differences in the configuration of the angle of the anterior chamber, it is shown that, apart from minor variations, there exists throughout the vertebrates an angular aqueous plexus or sinus (analogous to the canal of Schlemm in primate eyes) and that the bulk outflow of aqueous humour takes place across the mesothelial barrier of these aqueous plexuses via vacuolar transcellular channels in a manner exactly comparable to primates. Although an extensive analysis of arachnoid villi in sub-primate species has not been completed, preliminary studies indicate that macrovacuolar configurations may similarly be involved in the bulk outflow of CSF in a wide variety of vertebrates.
Isolated, perfused segments of late proximal straight tubule, descending thin limb of Henle, and ascending thick limb of Henle from the rabbit were studied before and after removal of the epithelium with sodium deoxycholate. The relationship between transmural hydrostatic pressure and outer tubule diameter was similar in paired intact tubules and basement membranes, indicating that basement membrane is the principal determinent of tubule distensibility. As calculated from teh measured perfusate flow at several different transmembrane hydrostatic pressures, the hydraulic conductivity of the basement membranes was 6-8 X 10(-3) cm3/cm2.min.cmH2O. With use of these LP values and the calculated oncotic pressure required experimentally to reduce transmembrane hydrostatic pressure transiently to zero, the apparent reflection coefficient of the basement membranes for serum albumin was estimated to be 0.05-0.16. It is concluded that basement membranes of the loop and of other previously studied segments of rabbit nephron provide very strong and elastic mechanical support to the epithelium while having minimal resistance to flow of water and of solutes as large as serum albumin.
The permeability of Necturus proximal tubule to hydrophilic nonelectrolytes of varying molecular size was studied under control conditions and during isotonic expansion of the animal's extracellular volume. Transepithelial permeability was measured in perfused tubular segments under conditions of zero net water flux. During volume expansion, tubular permeability to urea increased slightly, whereas mannitol decreased slightly and permeability to sucrose was significantly decreased. Volume expansion had a greater effect on osmotic flow parameters; the NaCl reflection coefficient decreased from 0.64 to 0.47 (summer animals) and from 0.41 to 0.27 (winter animals). Osmotic water flux and hydraulic conductivity increased but only in the lumen-to-capillary direction. Reflection coefficients of nonelectrolytes measured at the apical surface were reduced during volume expansion for probing molecules greater than 3 A in radius and were unchanged for smaller molecules, less than 3 A, suggesting two pore populations. We propose that an increase in tight-junction permeability can account for modification of osmotic flow parameters, whereas the whole thickness of the epithelium, particularly the intercellular space, plays the dominant role in regulation of diffusional permeability.
The scanning electron microscope was used to study the endothelium lining the trabecular wall of Schelemm's canal in 10 human eyes enucleated in treatment of choroidal melanomas. The eyes of 5 patients had been treated before enucleated with pilocarpine drops, and the remaining 5 were untreated and served as controls. In the pilocarpine-treated tissue there was an increased prominence of cellular bulges in the endothelial monolayer, and the endothelial pores were both larger and more frequent than in the untreated eyes. By the use of Poiseuille's formula it was calculated that the resistance offered by the pore system to the drainage of aqueous in the pilocarpine-treated group was approximately one-ninth of that in the control series.
The scanning electron microscope was used to compare the appearances of the endothelial monolayer of the trabecular wall of Schlemm's canal in the rhesus monkey after exposure to intracameral pressures of 8, 15 and 22 mm Hg for one hour.
Over this pressure range, the bulges in the spindle-shaped structures in the monolayer became rounder in shape and the number of openings on the surface was apparently greater at 22 mm Hg than at 15 and 8 mm Hg. A quantitative comparison between the tissue at 8 and 15 mm Hg showed a statistically significant increase in the number of openings on the bulges at 15 mm Hg.
Greater than 50 per cent of resistance to aqueous outflow was eliminated by internal trabeculotomy covering 30 degrees of the limbal circumference in enucleated, postmorten infant eyes subjected to constant-pressure acqueous perfusion. The proportion of total outflow resistance eliminated after limited trabeculotomy was greater in infant eyes than that observed in eyes of older children andthan that which others have observed in adult eyes. These data suggest that in the infant eye aqueous humor may flow circumferentially in Schlemm's canal but that the capacity for circumferential flow diminishes with increasing age.
The hydraulic flow conductivity of rabbit and bovine vitreous gel has been measured by a method that gives better results than the one previously used. The vitreous gel has a hydraulic flow conductivity that would be expected for a highly hydrated connective tissue. The "pore" size (the spaces availabel for water flow) are estimated to be about 4,000 A in diameter.
Morphological and physiological studies indicate multiple routes for transport across capillary endothelium. However, the identification of the morphological counterparts of specific transport processes (or the assignment of specific transport roles to morphologically identifiable pathways) has been only partly achieved: the contribution of endothelial cell membranes to transport of water and small, lipid-insoluble molecules needs to be evaluated. The identification of the "small pore" pathway for water and lipid-insoluble molecules with the intercellular junctions still remains questionable. The contributions to total macromolecular transport of junctions, single vesicles (pinocytosis, cytopempsis), chains of vesicles, and fenestrae are not yet known.
The effects of intraocular pressure on aqueous outflow facility and mechanical tension on the iridocorneal angle were evaluated in 44 excised postmortem human eyes. Mechanical tension on the iridocorneal angle was induced by depression of the crystalline lens posteriorly in a quantitative manner to simulate the effect of cyclotonia on the facility of aqueout outflow. In eyes perfused at constant intraocular pressure but with stepwise increases in lens depression, the increase in facility of outflow produced by the increased tension on the iridocorneal angle was minimal at low levels of intraocular pressure (2.5 and 5 mm Hg) and greatest at higher levels of intraocular pressure (10 to 25 mm Hg). Conversely, in pairs of eyes perfused over a graded series of increasing intraocular pressure, and subjected to maximal lens depression, facility of outflow remained constant until intraocular pressure exceeded 20 mm Hg; in the fellow eyes, perfused without lens depression, facility of outflow diminished linearly with increasing intraocular pressure.
A technique for measuring resistance to outflow in enucleated eyes is described. The technique minimizes the artifactual effects of ocular stretching and anterior chamber deepening. Using this perfusion technique, it was found that the resistance to outflow in normal, enucleated human eyes increases directly and linearly with intraocular pressure. This phenomenon, termed the outflow obstruction effect, was defined as the fractional increase in baseline outflow resistance (outflow resistance at an outflow pressure of zero) per millimeter of Hg intraocular pressure rise above baseline and was designated by the letter Q. The value of Q was found to be 0.012 mm. Hg-1 plus or minus 0.0014 indicating that the outflow resistance rise above its baseline value approximately 1 per cent per millimeter of Hg rise in intraocular pressure. Estimates of Q, calculated from data published by other investigators over the past two decades, are also included.
The nutrition of the intraocular tissues is accomplished by the retinal vessels, the uveal vessels, and by the aqueous humor. Both morphologically and physiologically the retinal vessels are similar to those in the brain. The endothelial cells of the capillaries are attached to each other by tight junctions, the resistance vessels respond poorly to a large number of drugs, and the blood flow through the retina is autoregulated and very little affected by the sympathetic nervous system. The blood vessels of the iris also have morphological and permeability characteristics similar to those in the brain but they are under a strong influence from the sympathetic nerves and react to many drugs. The blood flow is autoregulated. The blood vessels of the choroid and the ciliary processes are similar to those in the small intestine and in the kidney. The endothelial cells of the capillaries are fenestrated; the vessels respond to sympathetic nervous stimulation and to a large number of vasoactive drugs. Autoregulation of the blood flow is intermediate in the ciliary body and very poor or absent in the choroid...
Water rapidly crosses the plasma membrane of red blood cells (RBCs) and renal tubules through specialized channels. Although
selective for water, the molecular structure of these channels is unknown. The CHIP28 protein is an abundant integral membrane
protein in mammalian RBCs and renal proximal tubules and belongs to a family of membrane proteins with unknown functions.
Oocytes from Xenopus laevis microinjected with in vitro-transcribed CHIP28 RNA exhibited increased osmotic water permeability; this was reversibly inhibited
by mercuric chloride, a known inhibitor of water channels. Therefore it is likely that CHIP28 is a functional unit of membrane
water channels.
We tested the hypothesis that obstruction of the juxtacanalicular tissues, by melanin granules in pigmentary glaucoma and by other impermeable material in primary open angle glaucoma, leads to the development of a chronic glaucomatous condition. The distribution and concentration of melanin and other impermeable materials in the juxtacanalicular tissues and elsewhere in the trabecular meshwork was determined in 13 specimens. Six specimens were from patients with pigmentary glaucoma, two from patients with pigment dispersion syndrome, and three from patients with primary open angle glaucoma, as well as two from normal subjects. The effect of these materials on flow resistance was estimated using two hydrodynamic models. In model A, the electron-lucent spaces of the juxtacanalicular tissue were assumed to be open spaces, while in model B, these spaces and spaces filled with ground substance were assumed to be gel filled. In pigmentary glaucoma, 3.5% of the pigment was found in the juxtacanalicular tissue, while 96.5% was found in the corneoscleral and uveoscleral tissues. Permeabilities calculated according to model A were much higher than those expected from estimates of outflow facility in all groups, in agreement with the previous report of Ethier et al. The gel-filled spaces available for fluid flow, as determined by model B, showed no statistically demonstrable differences (pigmentary glaucoma, 32.9%; primary open angle glaucoma, 36.6%; pigment dispersion syndrome, 43.4%; normal, 44.1%). Furthermore, the amount of pigment present in the juxtacanalicular tissue was determined to have a negligible influence on permeability. Thus, the development of the chronic glaucomatous condition cannot be directly attributed to pigment accumulation in the juxtacanalicular tissue in pigmentary glaucoma.
A micropuncture technique involving the use of microcannulas with tip diameters less than 5 microns was used to measure the pressure in Schlemm's canal and in the meshwork at distances approximately 7 and 14 microns from the inner wall of Schlemm's canal. In one set of experiments where the spontaneous intraocular pressure (IOP) was 12.2 +/- 0.5 cmH2O and the Schlemm's canal pressure (PSc) was 7.6 +/- 0.7 cmH2O, the pressure at 7 microns from the inner wall of Schlemm's canal was found to be 8.9 +/- 0.7 cmH2O and at a distance of 14 microns, 11.0 +/- 0.5 cmH2O--that is, 1.3 +/- 0.2 and 3.4 +/- 0.3 cmH2O respectively, higher than the PSc. In another set of experiments, the spontaneous IOP and PSc were also measured and then the IOP was increased by means of an external reservoir and measured once again. Spontaneous IOP was 16.0 +/- 1.3 cmH2O and the PSc was 11.5 +/- 1.4 cmH2O before the IOP was increased. After the IOP was increased to 20.2 +/- 1.2 cmH2O, the PSc was 11.7 +/- 1.6 cmH2O. When the microcannula was introduced into the juxtacanalicular tissue to locations at about 7 and 14 microns from the inner wall of Schlemm's canal the pressure measured at 7 microns was 16.9 +/- 1.3 and at 14 microns it was 18.9 +/- 1.4 cmH2O--that is, 5.2 +/- 0.8 and 7.2 +/- 1.0 cmH2O respectively, higher than the PSc. The results indicate that at the spontaneous IOP about 75% of the resistance between the anterior chamber and Schlemm's canal is located within 14 microns from the canal with some 50% being located within the region 7 and 14 microns from the canal. After a small increase in IOP, the tissue causing most of the outflow resistance became relocated to a region within 7 microns from the canal.