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 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 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 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 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 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 A Porcine Ex Vivo Model of Pigmentary Glaucoma

2018 ◽  
Author(s):  
Yalong Dang ◽  
Susannah Waxman ◽  
Chao Wang ◽  
Ralitsa T. Loewen ◽  
Ming Sun ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Anterior Segment ◽  
Pigment Dispersion ◽  
Pigmentary Glaucoma ◽  
Ex Vivo Model ◽  
Trabecular Meshwork Cells ◽  
Gene Expression Microarrays ◽  
Expression Microarrays ◽  
And Behavior

Pigment dispersion can lead to pigmentary glaucoma, a poorly understood condition of younger myopic eyes with fluctuating high intraocular pressure. It has been difficult to investigate its pathogenesis without a model similar to human eyes in size and behavior. Here we present a porcine ex vivo model that recreates several features of pigmentary glaucoma, including intraocular hypertension, accumulation of pigment in the trabecular meshwork, and declining phagocytosis. We found that trabecular meshwork cells regulate outflow, form actin stress fibers, and have a decreased phagocytic activity. Gene expression microarrays and a pathway analysis of TM monolayers as well as ex vivo anterior segment perfusion cultures indicated that RhoA plays a central role in regulating the cytoskeleton, motility, and phagocytosis in the trabecular meshwork, providing new insights and targets to investigate in pigmentary glaucoma.

scholarly journals A Comparative Genome-wide Transcriptome Analysis of Glucocorticoid Responder and Non-Responder Primary Human Trabecular Meshwork Cells

2021 ◽  
Author(s):  
Kathirvel Kandasamy ◽  
Ravinarayanan Haribalaganesh ◽  
Ramasamy Krishnadas ◽  
Veerappan Muthukkaruppan ◽  
Colin E Willoughby ◽  
...  
Keyword(s):  
Ocular Hypertension ◽  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Anterior Segment ◽  
Dexamethasone Treatment ◽  
Ex Vivo Model ◽  
Trabecular Meshwork Cells ◽  
Genome Wide ◽  
Contralateral Eye ◽  
Genome Wide Gene Expression

The genome-wide gene expression analysis of primary human trabecular meshwork (HTM) cells with known glucocorticoid (GC) responsiveness was not reported earlier. Therefore, the purpose of this study was to investigate genes and pathways involved in the GC responsiveness in human trabecular meshwork (HTM) cells using RNA sequencing. A perfusion cultured human anterior segment ex vivo model was utilized to identify the induction of GC-induced ocular hypertension in one eye of a paired eyes after dexamethasone treatment based on the maximum intraocular pressure response and in the contralateral eye, HTM cells were isolated to classify GC-responder and non-responder cells. Some previously reported and unique genes and their associated pathways were identified in HTM cells in response to dexamethasone treatment versus vehicle control and more significantly in GC-responder and non-responder cells. This study will open up the possibility of identifying suitable molecular targets which have the potential to treat GC-induced ocular hypertension/glaucoma.

scholarly journals Iris Morphological Features in Patients with 360° Angle-Closure Neovascular Glaucoma: An Anterior Segment Optical Coherence Tomography Study

2018 ◽  
Vol 9 (3) ◽  
pp. 449-456 ◽  
Author(s):  
Yui Kobayashi ◽  
Shunsuke Nakakura ◽  
Etsuko Terao ◽  
Yuki Fujio ◽  
Kanae Matsuya ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Trabecular Meshwork ◽  
Pupil Diameter ◽  
Anterior Segment ◽  
Neovascular Glaucoma ◽  
Morphological Features ◽  
Angle Closure ◽  
Control Group ◽  
Optical Coherence ◽  
Swept Source

Purpose: To investigate iris morphological features in 360° angle-closure neovascular glaucoma (NVG) by swept-source anterior segment optical coherence tomography (ASOCT). Patients and Methods: In this retrospective, clinic-based, comparative study, 14 patients with 360° angle-closure NVG and 14 healthy age-matched control subjects were enrolled. All patients enrolled had no prior glaucoma surgery but underwent cataract surgery with intraocular lens implantation. Horizontal scanning images of swept-source ASOCT were analyzed using software calipers in temporal and nasal angle areas. The iris thickness at 1 and 2 mm from the pupil edge, iris length, trabecular meshwork length, peripheral anterior synechia (PAS) length, PAS height ratio (PAS length/trabecular meshwork length), and pupil diameter were measured. Results: Between the groups, there were no statistically significant differences in iris length, trabecular meshwork length, and pupil diameter (p > 0.05). However, the iris thickness was significantly reduced in the NVG group compared with the control group in the temporal and nasal areas (0.306 vs. 0.563 mm/0.326 vs. 0.645 mm at 1 mm, 0.278 vs. 0.523 mm/0.282 vs. 0.546 mm at 2 mm, respectively) (mean, all p < 0.001). In the NVG group, PAS height ratios were 1.55 ± 0.45 (mean ± standard deviation) (range, 0.58–2.30) and 1.55 ± 0.78 (range, 0.68–3.68) at the temporal and nasal angles, respectively. Conclusions: In patients with 360° angle-closure NVG, the iris thickness decreased to about 50% of that in healthy subjects, and the PAS length exceeded the trabecular meshwork length by about 1.5 times.

Sign in / Sign up

Export Citation Format

Share Document