Trabecular Meshwork Region Research Articles

Deletion of transcription factor AP-2β from the developing murine trabecular meshwork region leads to progressive glaucomatous changes.

Glaucoma is one of the leading causes of irreversible blindness and can result from abnormalities in anterior segment structures required for aqueous humor outflow, including the trabecular meshwork (TM) and Schlemm's canal (SC). Transcription factors such as AP-2β play critical roles in anterior segment development. Here, we show that the Mgp-Cre knock-in (Mgp-Cre.KI) mouse can be used to target the embryonic periocular mesenchyme giving rise to the TM and SC. Fate mapping of male and female mice indicates that AP-2β loss causes a decrease in iridocorneal angle cells derived from Mgp-Cre.KI-expressing populations compared to controls. Moreover, histological analyses revealed peripheral iridocorneal adhesions in AP-2β mutants that were accompanied by a decrease in expression of TM and SC markers, as observed using immunohistochemistry. In addition, rebound tonometry showed significantly higher intraocular pressure (IOP) that was correlated with a progressive significant loss of retinal ganglion cells, reduced retinal thickness, and reduced retinal function, as measured using an electroretinogram, in AP-2β mutants compared with controls, reflecting pathology described in late-stage glaucoma patients. Importantly, elevated IOP in AP-2β mutants was significantly reduced by treatment with latanoprost, a prostaglandin analog that increases unconventional outflow. These findings demonstrate that AP-2β is critical for TM and SC development, and that these mutant mice can serve as a model for understanding and treating progressive human primary angle-closure glaucoma.

ITGA8 positive cells in the conventional outflow tissue exhibit Schlemm's canal endothelial cell properties

AimsElevated intraocular pressure is primarily induced by the increased resistance of conventional outflow of aqueous humor. Dysfunction of the juxtacanalicular region of trabecular meshwork (TM) and Schlemm's canal (SC) endothelium, as the main conventional outflow tissue, have been implicated as the major reasons for the increased resistance. Integrins are widespread in these tissues, especially alpha8 integrin (ITGA8). We aim to investigate the properties of cells expressing ITGA8 in the conventional outflow tissue. Main methodsFluorescence in situ hybridization (FISH) and immunofluorescence (IF) were performed to detect the mRNA and protein levels of ITGA8 in human conventional outflow tissue. ITGA8-positive cells were isolated from the cultured human TM cells through a magnetic bead-based approach. Flow Cytometry was used to determine the purification efficiency. The expressions of TM and SC biomarkers and dexamethasone-induced myocilin secretion capacity of ITGA8-positive cells was assessed by Real-time PCR, IF and Western blot. A gel contraction assay was performed to evaluate contractility of ITGA8-positive cells after endothelin 1 treatment. Key findingsITGA8 was found with robust expression near the inner wall of SC endothelium. After purification, the proportion of ITGA8-positive cells were increased by about 10%. ITGA8-positive cells were identified with the properties as SC endothelial cells, such as more robust expressions of SC biomarkers, less dexamethasone-inducible myocilin expression, and stronger contractility. SignificanceThis study demonstrated that cells expressing ITGA8 in SC region possess more properties as SC endothelial cells. Our data implicate a crucial role of ITGA8 in aqueous humor (AH) outflow resistance regulation.

Level-Set Method for Image Analysis of Schlemm's Canal and Trabecular Meshwork.

PurposeTo evaluate different segmentation methods in analyzing Schlemm's canal (SC) and the trabecular meshwork (TM) in ultrasound biomicroscopy (UBM) images.MethodsTwenty-six healthy volunteers were recruited. The intraocular pressure (IOP) was measured while study subjects blew a trumpet. Images were obtained at different IOPs by 50-MHz UBM. ImageJ software and three segmentation methods—K-means, fuzzy C-means, and level set—were applied to segment the UBM images. The quantitative analysis of the TM-SC region was based on the segmentation results. The relative error and the interclass correlation coefficient (ICC) were used to quantify the accuracy and the repeatability of measurements. Pearson correlation analysis was conducted to evaluate the associations between the IOP and the TM and SC geometric measurements.ResultsA total of 104 UBM images were obtained. Among them, 84 were adequately clear to be segmented. The level-set method results had a higher similarity to ImageJ results than the other two methods. The ICC values of the level-set method were 0.97, 0.95, 0.9, and 0.57, respectively. Pearson correlation coefficients for the IOP to the SC area, SC perimeter, SC length, and TM width were −0.91, −0.72, −0.66, and −0.61 (P < 0.0001), respectively.ConclusionsThe level-set method showed better accuracy than the other two methods. Compared with manual methods, it can achieve similar precision, better repeatability, and greater efficiency. Therefore, the level-set method can be used for reliable UBM image segmentation.Translational RelevanceThe level-set method can be used to analyze TM and SC region in UBM images semiautomatically.

An In Vitro Bovine Cellular Model for Human Schlemm's Canal Endothelial Cells and Their Response to TGFβ Treatment.

PurposeDue to the limited availability of primary human Schlemm's canal (SC) endothelial cells, we aimed to develop an in vitro cellular model using the angular aqueous plexus (AAP) cells from bovine eyes.MethodsWe harvested a mixture of cells from the trabecular meshwork region including AAP loops from multiple donors, followed by puromycin treatment and immunostaining of Von Willebrand factor and vascular endothelial (VE)-cadherin to confirm identity. Previously identified differentially expressed genes in glaucomatous SC cells were examined in non-glaucomatous SC cells (n = 3) under 0% or 15% equibiaxial strain for 24 hours using droplet digital polymerase chain reaction (ddPCR) and analyzed using the Ingenuity Pathway Analysis (IPA) software application to identify upstream regulators. To compare the cellular responses to candidate regulators of these mechanoresponsive genes, AAP and human SC cells (n = 3) were treated with 5 or 10 ng/mL transforming growth factor beta-2 (TGFβ2) for 24 or 48 hours, followed with expression profiling using real-time PCR or ddPCR.ResultsWe found that the isolated AAP cells displayed uniform cobblestone-like morphology and positive expression of two endothelial markers. In stretched SC cells, nine glaucoma-related genes were upregulated, and IPA implicated TGFβ as a potential upstream regulator. The effects of TGFβ2 treatment were similar for both AAP and SC cells in a dose- and time-dependent manner, activating TGFBR1 and SMAD2, inhibiting BMP4, and altering expression of three glaucoma-related genes (DCN, EZR, and CYP1B1).ConclusionsBovine AAP cells may serve as an alternative cellular model of human SC cells.Translational RelevanceThese AAP cells may be used to study the functional pathways related to the outflow facility.

Noninvasive and Noncontact Sequential Imaging of the Iridocorneal Angle and the Cornea of the Eye.

PurposeHigh-resolution imaging of the critical anatomic structures of the eye, especially of the anterior chamber, in vivo, remains a challenge, even with currently available state-of-the-art medical imaging techniques. This study aims for the noninvasive and noncontact sequential imaging of the iridocorneal angle, especially the trabecular meshwork (TM) and the cornea of the eye in high-resolution using a newly developed imaging platform.MethodsBessel beam scanned light sheet fluorescence microscopy is used to attain high-resolution images of the TM. The ability of the Bessel beam to self-reconstruct around obstacles increases the image contrast at the TM region inside eye by reducing scattering and shadow artifacts. With minimal modifications, the excitation arm of the developed imaging system is adapted for noncontact, high-resolution corneal imaging.ResultsHigh-resolution images of the TM structures and cellular-level corneal structures are obtained in ex vivo porcine eyes, and subsequently in New Zealand white rabbit, in vivo. The spatial resolution of the developed system is 2.19 µm and has a noncontact working distance of 20 mm.ConclusionsA high-resolution imaging platform for noncontact sequential imaging of the TM and the cornea of the eye is developed. This imaging system is expected to be of potential interest in the evaluation and diagnosis of glaucoma and corneal diseases.Translational RelevanceThe developed prototype offers the plausibility of in vivo, noncontact, and high-resolution imaging of the iridocorneal angle and cornea of the eye that will aid clinicians in diagnosing open-angle glaucoma and corneal diseases better.

Lentiviral Vector-Mediated Expression of Exoenzyme C3 Transferase Lowers Intraocular Pressure in Monkeys.

Primary open-angle glaucoma (POAG) is considered a lifelong disease characterized by optic nerve deterioration and visual field damage. Although the disease progression can usually be controlled by lowering the intraocular pressure (IOP), therapeutic effects of current approaches do not last long. Gene therapy could be a promising method for persistent treatment of the disease. Our previous study demonstrated that gene transfer of exoenzyme C3 transferase (C3) to the trabecular meshwork (TM) to inhibit Rho GTPase (Rho), the upstream signal molecule of Rho-associated kinase (ROCK), resulted in lowered IOP in normal rodent eyes. In the present study, we show that the lentiviral vector (LV)-mediated C3 expression inactivates RhoA in human TM cells by ADP ribosylation, resulting indisruption of the actin cytoskeleton and altered cell morphology. In addition, intracameral delivery of the C3 vector to monkey eyes leads to persistently lowered IOP without obvious signs of inflammation. This is the first report of using a vector to transduce the TM of an alive non-human primate with a gene that alters cellular machinery and physiology. Our results in non-human primates support that LV-mediated C3 expression in the TM may have therapeutic potential forglaucoma, the leading cause of irreversible blindness in humans.

Human stem cells home to and repair laser-damaged trabecular meshwork in a mouse model

Glaucoma is the leading cause of irreversible vision loss, and reducing elevated intraocular pressure is currently the only effective clinical treatment. The trabecular meshwork is the main resistance site for aqueous outflow that maintains intraocular pressure. In this study, we transplanted human trabecular meshwork stem cells (TMSCs) intracamerally into mice that received laser photocoagulation over a 180° arc of the trabecular meshwork. TMSCs preferentially homed and integrated to the laser-damaged trabecular meshwork region and expressed differentiated cell markers at 2 and 4 weeks. Laser-induced inflammatory and fibrotic responses were prevented by TMSC transplantation with simultaneous ultrastructure and function restoration. Cell affinity and migration assays and elevated expression of CXCR4 and SDF1 in laser-treated mouse trabecular meshwork suggest that the CXCR4/SDF1 chemokine axis plays an important role in TMSC homing. Our results suggest that TMSCs may be a viable candidate for trabecular meshwork refunctionalization as a novel treatment for glaucoma.

Quantification of Pulse-Dependent Trabecular Meshwork Motion in Normal Humans Using Phase-Sensitive OCT.

PurposeThe purpose of this study was to characterize the pulsatile motion of trabecular meshwork (TM) in normal subjects and demonstrate its changes in accommodation with phase-sensitive optical coherence tomography (PhS-OCT).MethodsA new PhS-OCT laboratory prototype was designed to measure pulsatile TM motion in 13 healthy humans. Two sets of images were captured in 10 subjects, first with best corrective refraction and the other with an additional 3.0 diopters of accommodation. In each image, both maximum velocity (MV) and cumulative displacement (CD) in two selected regions of TM, the internal (IMV and ICD) and external (EMV and ECD) region, were measured.ResultsFor all parameters the intraclass correlation coefficient was >0.75. Neither MV nor CD was significantly different between eyes in individual subjects (PIMV = 0.967, PEMV = 0.391, PICD = 0.603, PECD = 0.482). In 26 eyes, with best corrective refraction, the EMV was higher than the IMV (23.9 ± 9.8 vs. 18.9 ± 8.08 μm/s; P = 0.0001), as was the ECD compared with the ICD (0.340 ±0.125 vs. 0.264 ± 0.111 μm; P = 0.000004). With accommodation, MV and CD significantly increased (PIMV = 0.0003, PEMV = 0.0003, PICD = 0.019, and PECD = 0.007), whereas MV and CD in the external region were still larger than those in the internal area (PEMV vs. IMV = 0.009, PECD vs. ICD = 0.023).ConclusionsThis study demonstrates the differences in TM motion between the internal and external regions of TM and displays its change with accommodation. The findings and good reproducibility suggest PhS-OCT helps to understand TM function in regulation of IOP, and, with further refinements, it may be useful in clinical management of glaucoma.

Knockout of tissue transglutaminase ameliorates TGFβ2-induced ocular hypertension: A novel therapeutic target for glaucoma?

Glaucoma is a vision threatening optic neuropathy that affects millions of people worldwide. In primary open angle, increased intraocular pressure (IOP) is the main risk factor for the development of this disease. Studies investigating the causes and mechanisms of increased IOP show fibrotic changes in the trabecular meshwork (TM) that are different from those of age-matched controls. Tissue transglutaminase (TGM2), an extracellular matrix (ECM) crosslinking enzyme, covalently crosslinks ECM proteins and causes excessive ECM protein deposition in the TM that could cause increased IOP. Previous literature reports increased expression of TGM2 in glaucomatous eyes compared to controls. We recently have shown that overexpression of TGM2 causes increased ECM crosslinking in the TM, increases IOP, and decreases aqueous humor (AH) outflow facility in mouse eyes. Therefore, we wanted to study the effect of TGM2 knockout (KO) on IOP in TGM2 floxed mice. Ad5.Cre transduction caused partial KO of TGM2, which decreased TGM2 expression in the TM region of mouse eyes. TGM2 KO significantly decreased IOP by itself and also in TGFβ2 induced ocular hypertensive mice. TGM2 KO also restores the outflow facility in TGFβ2 transduced eyes. Overall, TGM2 KO rescued the TGFβ2-induced ocular hypertensive phenotype. Thus, TGM2 may offer potential as a new therapeutic target for glaucoma.

Tissue Transglutaminase Elevates Intraocular Pressure in Mice.

Tissue transglutaminase (TGM2) is elevated in glaucomatous trabecular meshwork (TM) tissues. We investigated whether increased expression of TGM2 increases extracellular matrix crosslinking in the TM, thereby increasing aqueous humor outflow resistance and elevating intraocular pressure (IOP) in mouse eyes. GTM3, primary human GTM 125-05, and cultured mouse TM cells were transduced with adenovirus serotype 5 expressing human transglutaminase 2 (Ad5.TGM2; multiplicity of infection [MOI]-75) and fixed for immunocytochemistry. To test the effect on IOP in living eyes, Ad5.TGM2 was injected intravitreally into one eye of BALB/cJ (n = 18) or C57BL/6J mice (n = 9). The uninjected contralateral eye and Ad5.GFP served as controls. Daytime conscious IOPs were measured twice per week. Aqueous outflow facility (C) was measured by constant flow infusion on completion of IOP measurements. Immunohistochemistry was performed on BALB/cJ mouse eyes to study TGM2 expression and activity. The treatment of cultured TM cells with Ad5.TGM2 increased immunostaining of N-ε(γ-glutamyl) lysine crosslinks. Ad5.TGM2 injection significantly increased IOP in BALB/cJ (15.86 mm Hg [injected] vs. 10.70 mm Hg [control]) and in C57BL/6J mice (17.09 mm Hg [injected] vs. 12.01 mm Hg [control]). Mean aqueous outflow facility in the injected eyes of BALB/cJ (0.013 μL/min/mm Hg) and C57BL/6J mice (0.012 μL/min/mm Hg) was significantly lower than in the uninjected control eyes (BALB/cJ, 0.021 μL/min/mm Hg; C57BL/6J, 0.019 μL/min/mm Hg). The Ad5.TGM2 transduction of mouse eyes increased TGM2 expression in the TM region and increased N-ε(γ-glutamyl) lysine crosslinks. The increased expression of TGM2 in the TM increases N-ε(γ-glutamyl) lysine crosslinking in the TM, increases aqueous outflow resistance, and elevates IOP in mice. TGM2 may be at least partially responsible for ocular hypertension in POAG.

In vitro study of air bubble dynamics following pneumodissection of donor corneas and relationship of air bubble pattern with a peripheral paracentesis incision

AimsTo study various types of morphological patterns of the air bubble and their relation to a peripheral paracentesis after air injection in corneal stroma in vitro experiment.MethodsAir was injected into...

Anterior segment dysgenesis correlation with epithelial-mesenchymal transition in Smad4 knockout mice.

To explore the molecular mechanisms in lens development and the pathogenesis of Peters anomaly in Smad4 defective mice. Le-Cre transgenic mouse line was employed to inactivate Smad4 in the surface ectoderm selectively. Pathological techniques were used to reveal the morphological changes of the anterior segment in Smad4 defective eye. Immunohistochemical staining was employed to observe the expression of E-cadherin, N-cadherin and α-SMA in anterior segment of Smad4 defective mice and control mice at embryonic (E) day 16.5. Real-time quantitative polymerase chain reaction (qPCR) was performed to detect the expression of Snail, Zeb1, Zeb2 and Twist2 in lens of Smad4 defective mice and control mice at E16.5. Statistical evaluations were performed using the unpaired Student's t-test (two-tailed) by SPSS 11.0 software. Conditional deletion of Smad4 on eye surface ectoderm resulted in corneal dysplasia, iridocorneal angle closure, corneolenticular adhesions and cataract resembling Peters anomaly. Loss of Smad4 function inhibited E-cadherin expression in the lens epithelium cells and corneal epithelium cells in Smad4 defective eye. Expression of N-cadherin was up-regulated in corneal epithelium and corneal stroma. Both E-cadherin and N-cadherin were down-regulated at the future trabecular meshwork region in mutant eye. The qPCR results showed that the expression of Twist2 was increased significantly in the mutant lens (P<0.01). Smad4 is essential to eye development and likely a candidate pathogenic gene to Peters anomaly by regulating epithelial-mesenchymal transition. Twist2 can be regulated by Smad4 and plays an essential role in lens development.

Stem Cells in the Trabecular Meshwork for Regulating Intraocular Pressure.

Intraocular pressure (IOP) is still the main treatment target for glaucoma. Outflow resistance mainly exists at the trabecular meshwork (TM) outflow pathway, which is responsible for IOP regulation. Changes of TM cellularity and TM extracellular matrix turnover may play important roles in IOP regulation. In this article, we review basic anatomy and physiology of the outflow pathway and TM stem cell characteristics regarding the location, isolation, identification and function. TM stem cells are localized at the insert region of the TM and are label-retaining in vivo. They can be isolated by side-population cell sorting, cloning culture, or sphere culture. TM stem cells are multipotent with the ability to home to the TM region and differentiate into TM cells in vivo. Other stem cell types, such as adipose-derived stem cells, mesenchymal stem cells and induced pluripotent stem cells have been discovered for TM cell differentiation and TM regeneration. We also review glaucomatous animal models, which are suitable to study stem cell-based therapies for TM regeneration.

The Function of Matricellular Proteins in the Lamina Cribrosa and Trabecular Meshwork in Glaucoma.

To review the current literature regarding the role of matricellular proteins in glaucoma, specifically in the lamina cribrosa (LC) region of the optic nerve head (ONH) and the trabecular meshwork (TM). A literature search was performed for published articles describing the expression and function of matricellular proteins such as thrombospondin (TSP), connective tissue growth factor (CTGF), secreted protein acidic and rich in cysteine (SPARC), and periostin in glaucoma. In glaucoma, there are characteristic extracellular matrix (ECM) changes associated with optic disc cupping in the ONH and subsequent visual field defects. Matricellular proteins are a family of nonstructural secreted glycoproteins, which enable cells to communicate with their surrounding ECM, including CTGF, also known as CCN2, TSPs, SPARC, periostin, osteonectin, and tenascin-C and -X, and other ECM proteins. Such proteins appear to play a role in fibrosis and increased ECM deposition. Importantly, most are widely expressed in tissues particularly in the TM and ONH, and deficiency of TSP1 and SPARC has been shown to lower intraocular pressure in mouse models of glaucoma through enhanced outflow facility. This article highlights the role of matricellular proteins in glaucoma pathology. The potential role of these proteins in glaucoma is emerging as some have an association with the pathophysiology of the TM and LC region and might therefore be potential targets for therapeutic intervention in glaucoma.

Effect of persistent high intraocular pressure on microstructure and hydraulic permeability of trabecular meshwork**Project supported by the National Natural Science Foundation of China (Grant Nos. 31070840, 10802053, and 81201123), the Natural Science Foundation of Beijing, China (Grant No. 3122010), the Natural Science Foundation of Guangdong Province, China (Grant No. S2012040006576), the Shenzhen Science and Technology Innovation Committee, China (Grant No.

As the aqueous humor leaves the eye, it first passes through the trabecular meshwork (TM). Increased flow resistance in this region causes elevation of intraocular pressure (IOP), which leads to the occurrence of glaucoma. To quantitatively evaluate the effect of high IOP on the configuration and hydraulic permeability of the TM, second harmonic generation (SHG) microscopy was used to image the microstructures of the TM and adjacent tissues in control (normal) and high IOP conditions. Enucleated rabbit eyes were perfused at a pressure of 60 mmHg to achieve the high IOP. Through the anterior chamber of the eye, in situ images were obtained from different depths beneath the surface of the TM. Porosity and specific surface area of the TM in control and high IOP conditions were then calculated to estimate the effect of the high pressure on the permeability of tissue in different depths. We further photographed the histological sections of the TM and compared the in situ images. The following results were obtained in the control condition, where the region of depth was less than 55 μm with crossed branching beams and large pores in the superficial TM. The deeper meshwork is a silk-like tissue with abundant fluorescence separating the small size of pores. The total thickness of pathway tissues composed of TM and juxtacanalicular (JCT) is more than 100 μm. After putting a high pressure on the inner wall of the eye, the TM region progressively collapses and decreases to be less than 40 μm. Fibers of the TM became dense, and the porosity at 34 μm in the high IOP condition is comparable to that at 105 μm in the control condition. As a consequent result, the permeability of the superficial TM decreases rapidly from 120 μm2 to 49.6 μm2 and that of deeper TM decreases from 1.66 μm2 to 0.57 μm2. Heterogeneity reflected by descent in permeability reduces from 12.4 μm of the control condition to 3.74 μm of the high IOP condition. The persistently high IOP makes the TM region collapse from its normal state, in which the collagen fibers of the TM are arranged in regular to maintain the physiological permeability of the outflow pathway. In the scope of pathologically high IOP, the microstructure of the TM is sensitive to pressure and hydraulic permeability can be significantly affected by IOP.

The Effects of Tenascin C Knockdown on Trabecular Meshwork Outflow Resistance

Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP). Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy. TNC mRNA and protein levels were quantitated in anterior segments perfused at physiological and elevated pressure. Short, hairpin RNA (shRNA) silencing lentivirus targeting full-length TNC (shTNC) was applied to anterior segment perfusion organ cultures. The IOPs and central corneal thickness (CCT) of wild-type, TNC(-/-), and tenascin X (TNX(-/-)) knockout mice were measured. TNC was distributed in the juxtacanalicular (JCT) region of adult human TM, predominantly in the basement membrane underlying the inner wall of Schlemm's canal. Application of shTNC lentivirus to human and porcine anterior segments in perfusion culture did not significantly affect outflow rate. Although TNC was upregulated in response to pressure, there was no difference in outflow rate when shTNC-silenced anterior segments were subjected to elevated pressure. Furthermore, IOPs and CCTs were not significantly different between TNC(-/-) or TNX(-/-) and wild-type mice. TNC does not appear to contribute directly to outflow resistance. However, TNC immunolocalization in the JCT of adult human eyes suggests that certain areas of the TM are being continuously remodeled with or without an IOP increase.

Two-Photon Imaging of the Mouse Eye

To image the conventional aqueous outflow pathway and adjacent structures within the intact enucleated mouse eye using a noninvasive microscopy technique. Two-photon microscopy (2PM) techniques, including two-photon autofluorescence (2PAF) and second harmonic generation (SHG), were used to obtain images of the trabecular meshwork (TM) region within an intact mouse eye. Cardiac perfusion of fluorescein-conjugated dextran was used to label blood vessels within the eye to serve as an anatomic reference. Eyes were subsequently fixed, paraffin embedded, sectioned, and stained for comparison to the 2PM images. Three-dimensional analyses of multiple 2PM images revealed a well-defined region adjacent to the iris and cornea that is free of SHG signal and consistent with the location of Schlemm's canal. This open region is continuous with smaller tube structures consistent with collector channels. These structures do not label in mice perfused with the vascular probe dextran, supporting the hypothesis that the enclosed spaces are filled with aqueous humor rather than circulating blood. The TM region in the mouse eye was also visible, with a clear SHG signal representing collagen fibers. These results support the hypothesis that 2PM may be useful for noninvasively imaging the conventional aqueous outflow pathway in mouse models of glaucoma. Studies are ongoing to validate our methodology in live animals.

A rabbit model of age‐dependant ocular hypertensive response to topical corticosteroids

To investigate the ocular hypertensive response to topical dexamethasone (DEX), rimexolone (RIM), loteprednol etabonate (LOT) and fluorometholone (FML) in rabbits of different ages. Seventy-five rabbits of three age groups (7 weeks, 6 months and 1-year old) received topical administration of 0.1% DEX, 1% RIM, 0.5% LOT, 0.1% FML or balanced salt solution four times daily for 1 month. Intraocular pressure (IOP) was monitored at regular time intervals. After a month, eyes were harvested for histological study with haematoxylin and eosin (H&E), periodic acid Schiff and Masson trichrome staining. Trabecular meshwork changes were graded by masked ocular pathologists. Topical DEX caused the greatest increase in IOP, followed by RIM and FML. LOT caused the least IOP increase. Similar pattern of IOP response to the four corticosteroids was observed in the three studied age groups. Young rabbits (7 week) were the most responsive to corticosteroids among the age groups. Extracellular matrix thickening in the trabecular meshwork region and loss of trabecular meshwork cells were observed after DEX, FML or RIM treatments. Young rabbits are more susceptible to steroid induced increase in IOP, even for milder steroids such as fluorometholone and rimexolone.

A New Method for Selection of Angular Aqueous Plexus Cells from Porcine Eyes: A Model for Schlemm's Canal Endothelium

The authors sought to develop a technique for isolating and culturing angular aqueous plexus (AAP) cells from more plentiful porcine eyes. AAP is an analogue of Schlemm's canal. Cells were differentially selected with puromycin, a toxin often used to select brain microvascular endothelial cells based on the expression of P-glycoprotein (P-gp), a multidrug resistance efflux pump. Trabecular meshwork containing AAP was dissected and pooled from fresh porcine eyes, digested in collagenase I, washed, filtered, and cultured for 8 days in a gelatin-coated plastic flask. Cells were then selected by exposure to 4 μg/mL puromycin for 2 days in the culture medium. Cells were fixed and immunostained for P-gp, ICAM II, von Willebrand factor (vWF), VE-cadherin, and α-smooth muscle actin (α-SMA). Histology of the limbus showed that the dissection was limited to the trabecular meshwork region, including the AAP. Before puromycin treatment, cells appeared heterogeneous and polygonal, suggestive of a mixed population. More than 90% of the cells were removed by puromycin, leaving a population that appeared uniformly cobblestone-like when grown to confluence and that was contact inhibited. Puromycin-selected cells stained positively for the endothelial markers ICAM II, vWF, and VE-cadherin but negatively for α-SMA, consistent with staining patterns in whole tissue. Based on marker expression, morphology, and behavior in culture, puromycin-selected cells from porcine outflow tissues are AAP endothelial cells. Thus, porcine eyes can provide a plentiful alternative cell source for studying Schlemm's canal biology related to ocular hypertension.

Ultrastructure and Fluid Flow Physiology of Fetal Trabecular Meshwork Cells

Purpose: To assess the ultrastructural and fluid flow characteristics of cultured trabecular meshwork (TM) cells derived from fetal sources. Methods: Fetal eyes were carefully dissected to isolate the developing TM tissue for culture. Immunostaining was used to assess the expression of the junction-associated proteins zonula occludens-1 (ZO-1) and occludin. Fetal and adult TM cells were grown to confluence on permeable membranes for both flow and ultrastructural studies. Fluid flow resistance was measured by permeation of horseradish peroxidase (hrp) activity and hydraulic conductivity (HC) experiments. The effects of dexamethasone (Dex) on permeability and HC were also evaluated. Results: ZO-1 and occludin are expressed in the TM region of tissue sections and at cell borders in cultured fetal and adult TM cells. Transmission electron microscopy demonstrated that cultured TM cells possessed numerous mitochondria, electron-dense bodies, surface microvilli, and adherens and gap junctions. The permeation of hrp across fetal TM cell monolayers (0.030 ± 0.010) and of adult TM cells (0.031 ± 0.010) had similar values for absorbance at 470 nm (p = 0.83, 95% CI: −0.004, 0.005). Dex treatment significantly reduced the permeability to 0.022 ± 0.008 (p = 0.002) and 0.018 ± 0.009 (p = 0.004) for fetal and adult TM cells, respectively. The average HC (μ l/min/mmHg/cm2) of fetal cells (2.78 ± 1.03) and of the adult cells (2.15 ± 1.31) was not significantly different (p = 0.24, 95% CI: −1.01, 0.26). Dex treatment significantly reduced HC in both fetal (1.24 ± 0.72, p = 0.0004) and adult (1.29 ± 0.29, p = 0.00001) TM. Conclusions: Cultured fetal TM cells exhibited similar expression of junctional proteins and ultrastructural features as their adult counterparts. The permeability and HC of the fetal cells were similar to their older adult counterparts. Dex treatment induced increased fluid flow resistance in both cell types. These cells may serve as a source for in vitro studies of meshwork physiology.