scholarly journals Correction: Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork

PLoS ONE ◽  
2020 ◽  
Vol 15 (8) ◽  
pp. e0238408
Author(s):  
Ramesh B. Kasetti ◽  
Pinkal D. Patel ◽  
Prabhavathi Maddineni ◽  
Gulab S. Zode
Keyword(s):  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Segment Model

scholarly journals Ex-vivo cultured human corneoscleral segment model to study the effects of glaucoma factors on trabecular meshwork

PLoS ONE ◽  
2020 ◽  
Vol 15 (6) ◽  
pp. e0232111
Author(s):  
Ramesh B. Kasetti ◽  
Pinkal D. Patel ◽  
Prabhavathi Maddineni ◽  
Gulab S. Zode
Keyword(s):  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Segment Model

scholarly journals Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 174 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Priyal Shah ◽  
Ralitsa T. Loewen ◽  
...  
Keyword(s):  
Intraocular Pressure ◽  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Pigment Dispersion ◽  
Pigmentary Glaucoma ◽  
Fluorescent Microspheres ◽  
Phagocytic Capacity ◽  
Pressure Transducers ◽  
Intraocular Pressure Elevation ◽  
Iop Elevation

Background: Outflow regulation and phagocytosis are key functions of the trabecular meshwork (TM), but it is not clear how the two are related in secondary open angle glaucomas characterized by an increased particle load. We hypothesized that diminished TM phagocytosis is not the primary cause of early ocular hypertension and recreated pigment dispersion in a porcine ex vivo model. Methods: Sixteen porcine anterior chamber cultures received a continuous infusion of pigment granules (Pg), while 16 additional anterior chambers served as controls (C). Pressure transducers recorded the intraocular pressure (IOP). The phagocytic capacity of the trabecular meshwork was determined by fluorescent microspheres. Results: The baseline IOPs in Pg and C were similar (P=0.82). A significant IOP elevation occurred in Pg at 48, 120, and 180 hours (all P<0.01, compared to baseline). The pigment did not cause a reduction in TM phagocytosis at 48 hours, when the earliest IOP elevation occurred, but at 120 hours onward (P=0.001 compared to C). This reduction did not result in an additional IOP increase at 120 or 180 hours compared to the first IOP elevation at 48 hours (P>0.05). Conclusions: In this porcine model of pigmentary glaucoma, an IOP elevation occurs much earlier than when phagocytosis fails, suggesting that two separate mechanisms might be at work.

scholarly journals Impact of freeze-thaw cytoablation on aqueous outflow patterns in ex vivo anterior chamber perfusion cultures and whole eyes

F1000Research ◽  
2021 ◽  
Vol 10 ◽  
pp. 525
Author(s):  
Raoul Verma-Fuehring ◽  
Mohamad Dakroub ◽  
Alicja Strzalkowska ◽  
Piotr Strzalkowski ◽  
Hong Han ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Structural Changes ◽  
Ex Vivo ◽  
Anterior Segment ◽  
Large Change ◽  
Invasive Technique ◽  
Freeze Thaw ◽  
Before And After ◽  
Human Eyes ◽  
Porcine Eyes

Background: Porcine eyes have been widely used as ex vivo models in glaucoma research, as they share similar features with human eyes. Freeze-thawing is a non-invasive technique that has been used to obliterate living cells in anterior segment ex vivo cultures, to prepare them for further research such as cellular repopulation. This technique has previously been shown to reduce the intraocular pressure (IOP) in porcine eyes. The aim of this study was to investigate whether freeze-thaw cytoablation causes corresponding canalogram outflow changes in perfused anterior segment cultures (AFT) and whole porcine eyes (WFT). We hypothesized that the known IOP drop in AFT after trabecular meshwork ablation by freeze-thaw would be accompanied by a similarly large change in the distal outflow pattern. Methods: Two-dye (fluorescein and Texas red) reperfusion canalograms were used to compare the outflow time before and after two -80°C cycles of freeze-thaw. We assigned 28 freshly enucleated porcine eyes to four groups: perfused anterior segment dye controls (ACO, n = 6), perfused whole eye dye controls (WCO, n = 6), freeze-thaw treated anterior segment cultures (AFT, n = 10), and freeze-thaw treated whole eyes (WFT, n = 6). Results: In control groups ACO and WCO, the two different dyes had similar filling times. In AFT, the outflow pattern and filling times were unchanged. In WFT, the temporal superior quadrant filled more slowly (p = 0.042) while all others remained unchanged. The qualitative appearance of distal outflow spaces was altered only in some eyes. Conclusions: Freeze-thaw cytoablation caused neither loss nor leakage of distal outflow structures. Surprisingly, the loss of an intact trabecular meshwork over the entire circumference did not result in a general acceleration of quadrant outflow times. The results validate freeze-thawing as a method to generate an extracellular matrix without major structural changes.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

2017 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Suicide Gene ◽  
Control Group ◽  
Freeze Thaw

Objective: Trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might represent a novel therapeutic breakthrough. Various decellularized TM scaffolds were developed by ablating existing cells with suicide gene therapy or saponin, but always with incomplete cell removal or dissolve the extracellular matrix. We hypothesized that a chemical-free, freeze-thaw method would be able to produce a fully decellularized TM scaffold for cell transplantation. Materials and Methods: We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion and pressure transduction system within two hours of sacrifice. After they stabilized for 72 hours, eight eyes each were assigned to freeze-thaw (F) ablation (-80°C×2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The trabecular meshwork was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture medium for 180 hours. We assessed histological changes by hematoxylin and eosin staining. TM cell viability was evaluated with a calcein AM/propidium iodide (PI) assay. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with 5 degrees of freedom. Results: F and S experienced a similar IOP reduction by 30% from baseline (P=0.64). IOP reduction of about 30% occurred in F within 24 hours and in S within 48 hours. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to numerous PI-labeled dead TM cells and calcein-labeled viable TM cells in S. Conclusion: We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM excellular matrix in an organotypic perfusion culture.

scholarly journals Intraocular pressure elevation precedes a phagocytosis decline in a model of pigmentary glaucoma

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 174
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Priyal Shah ◽  
Ralitsa T. Loewen ◽  
...  
Keyword(s):  
Intraocular Pressure ◽  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Pigment Dispersion ◽  
Pigmentary Glaucoma ◽  
Fluorescent Microspheres ◽  
Phagocytic Capacity ◽  
Pressure Transducers ◽  
Intraocular Pressure Elevation ◽  
Iop Elevation

Background: Outflow regulation and phagocytosis are key functions of the trabecular meshwork (TM), but it is not clear how the two are related in secondary open angle glaucomas characterized by an increased particle load. We hypothesized that diminished TM phagocytosis is not the primary cause of early ocular hypertension and recreated pigment dispersion in a porcine ex vivo model. Methods: Sixteen porcine anterior chamber cultures received a continuous infusion of pigment granules (Pg), while 16 additional anterior chambers served as controls (C). Pressure transducers recorded the intraocular pressure (IOP). The phagocytic capacity of the trabecular meshwork was determined by fluorescent microspheres. Results: The baseline IOPs in Pg and C were similar (P=0.82). A significant IOP elevation occurred in Pg at 48, 120, and 180 hours (all P<0.01, compared to baseline). The pigment did not cause a reduction in TM phagocytosis at 48 hours when the earliest IOP elevation occurred, but at 120 hours onward (P=0.001 compared to C). This reduction did not result in an additional IOP increase at 120 or 180 hours compared to the first IOP elevation at 48 hours (P>0.05). Conclusions: In this porcine model of pigmentary glaucoma, an IOP elevation occurs much earlier than when phagocytosis fails, suggesting that two separate mechanisms might be at work.

scholarly journals Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells

2021 ◽  
Vol 118 (13) ◽  
pp. e2021942118
Author(s):  
Myoung Sup Shim ◽  
April Nettesheim ◽  
Angela Dixon ◽  
Paloma B. Liton
Keyword(s):  
Trabecular Meshwork ◽  
Primary Cilia ◽  
Molecular Mechanisms ◽  
Ex Vivo ◽  
Mechanical Stretch ◽  
Cell Types ◽  
Elevated Pressure ◽  
Compensatory Response ◽  
Protein Levels ◽  
Trabecular Meshwork Cells

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

scholarly journals Freeze-thaw decellularization of the trabecular meshwork in an ex vivo eye perfusion model

PeerJ ◽  
2017 ◽  
Vol 5 ◽  
pp. e3629 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Adrianna Jensen ◽  
Ralitsa T. Loewen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trabecular Meshwork ◽  
Degrees Of Freedom ◽  
Culture Media ◽  
Ex Vivo ◽  
Fluorescent Microscopy ◽  
Anterior Segment ◽  
Perfusion Culture ◽  
Control Group ◽  
Freeze Thaw

Objective The trabecular meshwork (TM) is the primary substrate of outflow resistance in glaucomatous eyes. Repopulating diseased TM with fresh, functional TM cells might be a viable therapeutic approach. Decellularized TM scaffolds have previously been produced by ablating cells with suicide gene therapy or saponin, which risks incomplete cell removal or dissolution of the extracellular matrix, respectively. We hypothesized that improved trabecular meshwork cell ablation would result from freeze-thaw cycles compared to chemical treatment. Materials and Methods We obtained 24 porcine eyes from a local abattoir, dissected and mounted them in an anterior segment perfusion within two hours of sacrifice. Intraocular pressure (IOP) was recorded continuously by a pressure transducer system. After 72 h of IOP stabilization, eight eyes were assigned to freeze-thaw (F) ablation (−80 °C × 2), to 0.02% saponin (S) treatment, or the control group (C), respectively. The TM was transduced with an eGFP expressing feline immunodeficiency viral (FIV) vector and tracked via fluorescent microscopy to confirm ablation. Following treatment, the eyes were perfused with standard tissue culture media for 180 h. TM histology was assessed by hematoxylin and eosin staining. TM viability was evaluated by a calcein AM/propidium iodide (PI) assay. The TM extracellular matrix was stained with Picro Sirius Red. We measured IOP and modeled it with a linear mixed effects model using a B-spline function of time with five degrees of freedom. Results F and S experienced a similar IOP reduction of 30% from baseline (P = 0.64). IOP reduction of about 30% occurred in F within 24 h and in S within 48 h. Live visualization of eGFP demonstrated that F conferred a complete ablation of all TM cells and only a partial ablation in S. Histological analysis and Picro Sirius staining confirmed that no TM cells survived in F while the extracellular matrix remained. The viability assay showed very low PI and no calcein staining in F in contrast to many PI-labeled, dead TM cells and calcein-labeled viable TM cells in S. Conclusion We developed a rapid TM ablation method that uses cyclic freezing that is free of biological or chemical agents and able to produce a decellularized TM scaffold with preserved TM extracellular matrix in an organotypic perfusion culture.

scholarly journals A porcine ex vivo model of pigmentary glaucoma

2017 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Ralista T. Loewen ◽  
Ming Sun ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Pigment Dispersion ◽  
Pigmentary Glaucoma ◽  
Ex Vivo Model ◽  
Pigment Dispersion Syndrome ◽  
And Behavior ◽  
Human Eyes ◽  
Pigment Accumulation

AbstractPigment dispersion syndrome can lead to pigmentary glaucoma (PG), a poorly understood condition of younger, myopic eyes with fluctuating, high intraocular pressure (IOP). The absence of a model similar in size and behavior to human eyes has made it difficult to investigate its pathogenesis. Here, we present a porcine ex vivo model that recreates the features of PG including intraocular hypertension, pigment accumulation in the trabecular meshwork and relative failure of phagocytosis. Inin vitromonolayer cultures as well as inex vivoeye perfusion cultures, we found that the trabecular meshwork (TM) cells that regulate outflow, form actin stress fibers and have a decreased phagocytosis. Gene expression microarray and pathway analysis indicated key roles of RhoA in regulating the TM cytoskeleton, motility, and phagocytosis thereby providing new targets for PG therapy.

Sign in / Sign up

Export Citation Format

Share Document