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Comparison of results of atomic force microscopy (AFM), confocal microscopy, light microscopy, and transmission electron microscopy (TEM) between eyes with experimental glaucoma (ExGl/OD) and untreated control (OS) eyes (n = 8 animals for AFM; n = 3 animals for microscopy). (A) Box and whisker plots demonstrating the relative elastic moduli of untreated normal eyes (blue) and eyes with ExGl (red). (B) Comparison of the trabecular meshwork (TM) thickness between eyes with ExGl and normal control eyes. (i-ii): Representative confocal images show that the TM thickness (double arrow) was smaller in a high-tracer region of an eye with ExGl (i) compared to similar region of a normal eye (ii). SC = Schlemm's canal. (iii): The TM thickness was significantly smaller in the high-tracer regions of eyes with ExGl compared to similar regions of the normal eyes. *: p < 0.05. Fluorescence of the tracer dye is indicated in red. (C) Fluorescence Intensity (i-ii) Representative confocal images showed no significant difference in (iii) fluorescence intensity between non-lasered regions of ExGl eyes and similar region of normal control eyes. (D) Height of Schlemm's canal (SC). (i-ii): Representative light microcopy images showed that the (iii) height of SC was significantly narrower
Source publication
Laser-induced experimental glaucoma (ExGl) in non-human primates (NHPs) is a common animal model for ocular drug development. While many features of human hypertensive glaucoma are replicated in this model, structural and functional changes in the unlasered portions of trabecular meshwork (TM) of laser-treated primate eyes are understudied. We stud...
Contexts in source publication
Context 1
... Force Microscopy (AFM). We investigated if there were any correlation between elevated IOP and TM stiffness. The elastic moduli of the TM from both eyes of 6 animals (Set B) are shown in Fig. 6A. Elastic mod- uli were also determined for 2 animals from Set C. The mean elastic moduli of the control TM was 3.31 ± 0.32 kPa for Set B and 2.63 ± 0.14 kPa for two animals from Set C. Mean elastic moduli for the unlasered regions of TM in all eyes with ExGl were approximately 6-fold lower (0.464 ± 0.036 kPa for Set B, and 0.151 ± ...
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... meshwork thickness. TM thickness, measured in confocal images, was found to be significantly less in the high-tracer regions of eyes with ExGl (i.e. unlasered region of ExGl eyes) compared to similar regions of the normal eyes (mean 53.7 ± 3.1 vs. 127.4 ± 24.0 μm, p = 0.03) (Fig. ...
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... While there was visually a trend towards greater amounts of tracers in the high-flow regions of the eyes with ExGl compared to normal eyes by examining the confocal microscopy images; this difference was not statistically significant as quantified by fluorescence intensity measurement (71.6 ± 6.3 in ExGL vs 57.4 ± 8.9 in normal eyes, p = 0.2 (Fig. ...
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... of SC. The height of SC was significantly narrower or collapsed in some regions in eyes with ExGl com- pared to normal eyes. Mean SC height (the distance between the inner and outer walls of SC) was 12.3 ± 2.2 µm in ExGl compared to 25.0 ± 1.8 µm in normal eyes, p < 0.001 (Fig. ...
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... SC was analyzed ranged from 0.92-1.73 mm (the length being small due to only 1 clock-hour of tissue segment being left non-lasered in the eyes with ExGl). Significantly more giant vacuoles were found in the high-tracer regions of the normal eyes compared to those with high tracer regions with ExGl (mean 47 ± 9 vs 11 ± 8 GVs per mm SC, p < 0.01) (Fig. ...
Context 6
... tissue (JCT). By TEM, similar mor- phology was observed in the high-tracer regions of the JCT of both normal eyes and those with ExGl (i.e corre- sponding to unlasered region of ExGl eyes), where the JCT was loose and expanded. More inner wall endothelial cells of SC appeared to be lost in the eyes with ExGl compared to the normal eyes (Fig. ...
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Significance
Glaucoma is the leading cause of irreversible blindness worldwide. The primary and only modifiable risk factor for the development of glaucoma is elevated intraocular pressure (IOP), and lowering IOP effectively slows glaucomatous disease progression. Unfortunately, the majority of available treatments do not target, or intentionally b...
Citations
... The TM not only became thinner, but the structure became compact due to collapse. There have been many previous studies on the structure of TM in POAG, but most of the studies have been based on glucocorticoids, transforming growth factor (TGF)-b2, lasers, and other methods to model rodents, [36][37][38] and their consistency with spontaneous POAG is difficult to verify. Previous studies have suggested that TM tissue becomes thinner when POAG occurs; however our study found that thinning of the TM does not simply reduce the tissue component but becomes compact caused by collapsed tissue structure. ...
As the major channel of aqueous humor outflow, dysfunction of trabecular meshwork (TM) can lead to intraocular pressure elevating, which can trigger primary open-angle glaucoma (POAG). In this study, we use single-cell RNA sequencing (scRNA-seq) technique to build an atlas and further explore the spontaneous POAG and healthy macaques cellular heterogeneity associated with the dysfunction of TM contraction. We built the TM atlas, which identified 14 different cell types. In Beam A, Beam B, Beam C, and smooth muscle cell (SMC) cell types, we first found multiple genes associated with TM contraction (e.g., TPM1, ACTC1, TNNT1), determining their differential expression in the POAG and healthy groups. In addition, the microstructural alterations in TM of POAG non-human primates were observed, which was compact and collapsed. Thus, our study indicated that TPM1 may be a key target for regulating TM structure, contraction function, and resistance of aqueous humor outflow.
... In this study, median IOP was significantly greater in laser-treated (Miyahara et al., 2003;Raghunathan et al., 2017). While IOP in one animal did not reach persistently elevated levels following laser-treatment (Animal 6, OS), outflow facility was significantly decreased in this eye (0.04 μL/min/mmHg) and the eye displayed morphological changes similar to all other laser-treated eyes, therefore it was included in analysis. ...
Though roughly 30–50% of aqueous outflow resistance resides distal to Schlemm's canal (SC), the morphology of the conventional outflow pathway distal to SC has not been thoroughly evaluated. This study examined the morphological changes along proximal and distal aspects of the conventional aqueous outflow pathway and their association with decreased outflow facility in an experimental model of glaucoma in cynomolgus macaques. Nd:YAG laser burns were made to 270–340 degrees of the trabecular meshwork (TM) of one eye (n = 6) or both eyes (n = 2) of each monkey to induce ocular hypertension. Distinct regions of the TM were left unlasered. Contralateral eyes (n = 5) were not lasered and were utilized as controls. Monkeys were sacrificed ≥60 months after their last laser treatment. All eyes were enucleated and perfused at 15 mmHg for 30 min to measure outflow facility. Two pairs of eyes were also perfused with fluorescein to examine segmental outflow. All eyes underwent perfusion-fixation for 1 h. Anterior segments were cut into radial wedges and processed for light and electron microscopy. Width, height, and cross-sectional area (CSA) of SC were compared between high- and low-flow regions of control eyes, and between non-lasered regions of laser-treated eyes and control eyes. Number and CSA of intrascleral veins (ISVs) were compared between non-lasered and lasered regions of laser-treated eyes and control eyes, and between high- and low-flow regions of control eyes. Scleral collagen fibril diameter was compared between control eyes and lasered and non-lasered regions of laser-treated eyes. Median outflow facility was significantly decreased in laser-treated eyes compared to control eyes (P = 0.02). Median CSA and height of SC were smaller in high-flow regions than low-flow regions of control eyes (P < 0.05). Median width of SC was not significantly different between high- and low-flow regions of control eyes (P > 0.05). Median CSA, width, and height of SC were not different between non-lasered regions and control eyes (P > 0.05). SC was partially or completely obliterated in lasered regions. Median number of ISVs was significantly decreased in lasered regions compared to non-lasered regions (P < 0.01) and control eyes (P < 0.01). Median CSA of ISVs did not differ between these groups (P > 0.05). Median number and CSA of ISVs were not significantly different between high- and low-flow regions of control eyes (P > 0.05). Lasered regions displayed looser scleral stroma and smaller median diameter of collagen fibrils adjacent to the TM compared to non-lasered regions (P < 0.05) and control eyes (P < 0.05). Dense TM, partial to complete obliteration of SC, and a decreased number of patent ISVs may account in part for the decreased outflow facility in monkey eyes with laser-induced ocular hypertension. The significance of changes in scleral structure in laser-treated eyes warrants further investigation.
... In addition, ocular toxicity may be associated with IOP changes during the preclinical study. NHPs are superior model for ocular research due to its morphological and physiological similarities with humans [9,12]. All drugs which are clinical prescribed against glaucoma have been found to reduce the IOP in NHPs. ...
... In this study, we examined the IOP to compare the effects of four different anesthetics in male and female Vietnamese cynomolgus monkeys using rebound tonometer, TonoVet™. As cynomolgus monkeys can be a representative ocular disease model and are highly suitable for drug screening for glaucoma [12], we have utilized this strain for IOP measurement. TonoVet™ was validated as an accurate equipment for IOP measurement due to the high correlation with the actual eye pressure [18]. ...
Background
Nonhuman primates (NHPs) are superior model for ocular research due to its morphological and physiological similarities with humans. Thus, the effect of four different anesthetic combinations [ketamine (10 mg/kg), ketamine + xylazine (7 + 0.6 mg/kg), zoletil (4 mg/kg), and zoletil + xylazine (4 + 0.2 mg/kg)] on intraocular pressure (IOP) was determined in cynomolgus monkeys.
Results
The administration of ketamine + xylazine or zoletil + xylazine resulted in lower IOP compared to ketamine or zoletil alone. Moreover, the IOP in male monkeys was higher than in females. The difference between the right and left eye was not found.
Conclusions
Anesthetics affected the IOP, and gender differences should be considered when measuring the IOP of nonhuman primates (NHPs).
... One of the major factors implicated in the pathobiology of the TM associated with increased restriction to aqueous humor and elevated IOP in POAG is aberrant remodeling of the ECM. 13,14,21,[64][65][66] As a part of its diverse normal homeostatic functions, the ECM regulates and sequesters growth or bioactive factors from the extracellular milieu. 67,68 However, this ECM-dependent regulation and sequestration of bioactive factors is impaired in the event of aberrant ECM remod-eling. ...
PURPOSE. Lysophosphatidic acid (LPA) and soluble interleukin-6 receptor (sIL6R) are elevated in primary open angle glaucoma (POAG). LPA and IL6 modulate in response to biomechanical stimuli and converge on similar fibrotic phenotypes. Thus, we determined whether LPA and IL6 trans-signaling (IL6/sIL6R) interact via Yes-associated protein (YAP)/Transcriptional coactivator with a PDZ-binding motif (TAZ) or Signal transducer and activator of transcription 3 (STAT3) pathways in human trabecular meshwork (hTM) cells.
METHODS. Confluent primary hTM cells were serum starved for 24 hours, and treated with vehicle, LPA (20 μM), IL6 (100 ng/mL)/sIL6R (200 ng/mL), or both (LPA + IL6/sIL6R) for 24 hours, with or without a YAP inhibitor (verteporfin; 2 μM) or STAT3 inhibitor (2 μM). Expression of key receptors and ligands, signaling mediators, actomyosin machinery, cell contractility, and extracellular matrix (ECM) targets of both signaling pathways was determined by immunocytochemistry, RT-qPCR, and Western blotting.
RESULTS. LPA and IL6 trans-signaling coupling overexpressed/activated receptors and ligands, glycoprotein-130, IL6, and autotaxin; signaling mediators, YAP, TAZ, Pan-TEAD, and phosphorylated STAT3 (pSTAT3); actomyosin and contractile machinery components, myosin light chain 2 (MLC2), phosphorylated MLC2, rho-associated protein kinase 1, fila-mentous actin, and α-smooth muscle actin; and fibrotic ECM proteins, collagen I and IV, fibronectin, laminin, cysteine-rich angiogenic inducer 61, and connective tissue growth factor in hTM cells; mostly beyond LPA or IL6 trans-signaling alone. Verteporfin inhibited YAP, TAZ, and pSTAT3, with concomitant abrogation of aforementioned fibrotic targets; the STAT3 inhibitor was only partially effective.
CONCLUSIONS. These data suggest synergistic crosstalk between LPA and IL6 trans-signaling, mediated by YAP, TAZ, and pSTAT3. By completely inhibiting these mediators , verteporfin may be more efficacious in ameliorating LPA and/or IL6 trans-signaling-induced ocular hypertensive phenotypes in hTM cells.
... Although TM changes implicated in ocular hypertension are multifactorial, [9][10][11][12] aberrant TM extracellular matrix (ECM) remodeling is considered a primary contributory factor. 8,[13][14][15][16][17][18][19][20] One major component of TM ECM changes implicated in ocular hypertension and glaucoma is the increased expression and activity of ECM crosslinking enzymes like lysyl oxidase (LOX), LOX-like 1 to 4, and tissue transglutaminase 2 (TGM2). [21][22][23][24][25][26] For example, Raychaudhuri and colleagues 23 showed overexpression of TGM2, to induce crosslinks in the TM ECM, caused ocular hypertension in mice; conversely, TGM2-knockout mice had markedly reduced ocular hypertension. ...
PURPOSE. The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabec-ular meshwork (hTM) cells by dysregulating β-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways.
METHODS. Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/β-catenin and YAP/TAZ pathways were determined.
RESULTS. At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold (P < 0.001) stiffer, total β-catenin was unchanged, pβ-catenin was elevated, and pGSK3β was suppressed. Although 10% XCDM had no effect on cytoplasmic β-catenin levels, it reduced nuclear β-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear β-catenin.
CONCLUSIONS. Increased cytoplasmic TAZ may inhibit β-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplas-mic localization may be an important downstream event of how increased TM extracel-lular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.
... While the iris and cornea are well defined by the 5th month of gestation in humans, the angle undergoes additional maturation to clearly separate the TM from the ciliary body and iris root (Anderson, 1981;Cvekl and Tamm, 2004). At birth, the TM is soft (Raghunathan et al., 2017) and the corneoscleral meshwork beams are defined, but there is little space between the beams and their collagenous core is not apparent. Thus, the TM appears to be a continuous tissue mass (Anderson, 1981). ...
... We showed that unlasered regions of the TM in experimental glaucoma (ExGl) in non-human primates (NHPs) were softer compared with control unlasered NHPs (3.3 ± 0.32 kPa). This suggests that eyes (non-diseased) have the capacity to compensate for chronic IOP elevation in ExGl by altering the composition and subsequent mechanical properties of the ECM in the JCT region to compensate for reduced outflow facility (Raghunathan et al., 2017), although homeostasis may still be disrupted. In a follow up study , we compared elastic moduli in segmental flow regions of glaucomatous eyes where homeostatic response was lacking. ...
Glaucoma remains only partially understood, particularly at the level of intraocular pressure (IOP) regulation. Trabecular meshwork (TM) and Schlemm's canal inner wall endothelium (SCE) are key to IOP regulation and their characteristics and behavior are the focus of much investigation, thus, are becoming more apparent with time. We and others have studied the TM and SCE's extracellular matrix (ECM) extensively and unraveled much about its functions and role in regulating aqueous outflow. Ongoing ECM turnover is required to maintain IOP regulation and several TM ECM manipulations modulate outflow facility.
We have established clearly that the outflow pathway senses sustained pressure deviations and responds by adjusting the outflow resistance correctively to keep IOP within an appropriately narrow range which will not normally damage the optic nerve. The glaucomatous# outflow pathway has in many cases lost this IOP homeostatic response, apparently due, at least in part to loss of TM cells. Depletion of TM cells eliminates the IOP homeostatic response, while restoration of TM cells restores it. Aqueous outflow is not homogeneous, but rather segmental with regions of high, intermediate and low flow. In general, glaucomatous eyes have more low flow regions than normal eyes. There are distinctive molecular differences between high and low flow regions, and during the response to an IOP homeostatic pressure challenge, additional changes in segmental molecular composition occur. In conjunction with these changes, the biomechanical properties of the juxtacanalicular (JCT) segmental regions are different, with low flow regions being stiffer than high flow regions. The JCT ECM of glaucomatous eyes is around 20 times than in normal eyes.
The aqueous humor outflow resistance has been studied extensively, but neither the exact molecular components that comprise the resistance nor their exact location have been established. Our hypothetical model, based on considerable available data, posits that the continuous SCE basal lamina, which lies between 125 and 500 nm beneath the SCE basal surface, is the primary source of normal resistance. On the surface of JCT cells, small and highly controlled focal degradation of its components by Podosome- or invadopodia-like structures, PILS occurs in response to pressure-induced mechanical stretching. Sub-micron sized basement membrane discontinuities develop in the SCE basement membrane and these discontinuities allow passage of aqueous humor to and through SCE giant vacuoles and pores. JCT cells then relocate versican with its highly charged glycosaminoglycan side chains into the discontinuities and by manipulation of their orientation and concentration, the JCT and perhaps the SCE cells regulate the amount of fluid passage. Testing this outflow resistance hypothesis is ongoing in our lab and has the potential to advance our understanding of IOP regulation and of glaucoma.
... Any of these intrinsic stimuli can either act alone or in concert with other factors at any particular point in time to regulate a vast majority of behavioral biological responses of cells, be it biochemical and/or biomechanical. Further, dynamic remodeling of the ECM is a critical player in both homeostasis and disease (Acott et al., 2014;Raghunathan, Eaton, et al., 2017;Vranka et al., 2018), and thus has garnered much attention in biomedical research. Considering that the interactions between cells and tissues are bidirectional, an effect on the former has direct consequences on the latter and vice versa Vranka, Kelley, Acott, & Keller, 2015). ...
... Any of these intrinsic stimuli can either act alone or in concert with other factors at any particular point in time to regulate a vast majority of behavioral biological responses of cells, be it biochemical and/or biomechanical. Further, dynamic remodeling of the ECM is a critical player in both homeostasis and disease (Acott et al., 2014;Raghunathan, Eaton, et al., 2017;Vranka et al., 2018), and thus has garnered much attention in biomedical research. Considering that the interactions between cells and tissues are bidirectional, an effect on the former has direct consequences on the latter and vice versa Vranka, Kelley, Acott, & Keller, 2015). ...
Ocular hypertension has been attributed to increased resistance to aqueous outflow often as a result of changes in trabecular meshwork (TM) extracellular matrix (ECM) using in vivo animal models (for example, by genetic manipulation) and ex vivo anterior segment perfusion organ cultures. These are however complex and difficult in dissecting molecular mechanisms and interactions. In vitro approaches to mimic the underlying substrate exist by manipulating either ECM topography, mechanics, or chemistry. These models best investigate the role of individual ECM protein(s) and/or substrate property, and thus do not recapitulate the multifactorial extracellular microenvironment; hence, mitigating its physiological relevance for mechanistic studies. Cell-derived matrices (CDMs) however, are capable of presenting a 3D-microenvironment rich in topography, chemistry, and whose mechanics can be tuned to better represent the network of native ECM constituents in vivo. Critically, the composition of CDMs may also be fine-tuned by addition of small molecules or relevant bioactive factors to mimic homeostasis or pathology. Here, we first provide a streamlined protocol for generating CDMs from TM cell cultures from normal or glaucomatous donor tissues. Second, we document how TM cells can be pharmacologically manipulated to obtain glucocorticoid-induced CDMs and how generated pristine CDMs can be crosslinked with reagents like genipin. Finally, we summarize how CDMs may be used in mechanistic studies and discuss their probable application in future TM regenerative studies.
... Any of these intrinsic stimuli can either act alone or in concert with other factors at any particular point in time to regulate a vast majority of behavioral biological responses of cells, be it biochemical and/or biomechanical. Further, dynamic remodeling of the ECM is a critical player in both homeostasis and disease (Acott et al., 2014;Raghunathan, Eaton, et al., 2017;Vranka et al., 2018), and thus has garnered much attention in biomedical research. Considering that the interactions between cells and tissues are bidirectional, an effect on the former has direct consequences on the latter and vice versa Vranka, Kelley, Acott, & Keller, 2015). ...
Native extracellular matrix (ECM) based scaffolds are far more superior in structural and compositional complexity than other engineered scaffolding materials such as hydrogels, electrospun fibers, and three-dimensional (3D) printed substrates. Due to the presence of native structural proteins and other macromolecules, native ECM can better restore the crucial cell-ECM crosstalk and provide a highly biomimetic microenvironment to cells. Allogenic or xenogeneic tissues have been derived by decellularization to obtain native ECM scaffolds. However, their applicability is limited by batch to batch variation, risk of pathogen transfer, undesirable immune response and scarcity of donors. Human dermal fibroblasts (hDFs) can be prescreened and maintained in a pathogen-free condition. Herein, we have described a step-by-step protocol to generate a completely biological ECM scaffold by decellularization of hDF cell sheets. Decellularization was achieved by using an anionic surfactant sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetate (EDTA). The resulting ECM sheet was organized into a nanofibrous scaffold, containing major ECM structural proteins as well as other macromolecules including collagens, fibronectin, laminin and elastin. This cell-derived nanofibrous ECM is a promising scaffold material for constructing highly biomimetic functional tissues.
... Due to their evolutionary relatedness to human, non-human primates are currently used as animal models in a wide range of medical fields including infection biology [6], development of medical prosthetics [7], neuroscience [8], safety testing of pharmaceutical molecules [9], ophthalmology [10], vaccinology [11] and xenotransplantation [12], among others. ...
We are currently living the advent of a new age for medicine in which basic research is being quickly translated into marketable drugs, and the widespread access to genomics data is allowing the design and implementation of personalized solutions to medical conditions. Non-human primates (NHP) have gained an essential role in drug discovery and safety testing due to their close phylogenetic relationship to humans. In this study, a collection of well characterized genes of the human immune system was used to define the orthology-based immunome in four NHP species, with carefully curated annotations available based on multi-tissue RNA-seq datasets. A broad variation in the frequency of expressed protein isoforms was observed between species. Finally, this analysis also revealed the lack of expression of at least four different chemokines in new-world primates. In addition, transcripts corresponding to four genes including interleukin 12 subunit alpha were expressed in humans but no other primate species analyzed. Access to the non-human primate immunome is available in http://www.fidic.org.co:90/proyecto/.
