scholarly journals Trabecular Meshwork Motion Profile from Pulsatile Pressure Transients: A New Platform to Simulate Transitory Responses in Humans and Nonhuman Primates

2021 ◽  
Vol 12 (1) ◽  
pp. 11
Author(s):  
Chen Xin ◽  
Xiaofei Wang ◽  
Ningli Wang ◽  
Ruikang Wang ◽  
Murray Johnstone
Keyword(s):  
Time Constant ◽  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Pulse Amplitude ◽  
Relaxation Curve ◽  
Posterior Chamber ◽  
Transient Pressure ◽  
Motion Responses ◽  
Pulsatile Pressure ◽  
Mean Pressure

Trabecular meshwork (TM) motion abnormality is the leading cause of glaucoma. With technique limitations, how TM moves is still an enigma. This study describes a new laboratory platform to investigate TM motion responses to ocular transients in ex vivo eyes. The anterior segments of human cadaver and primate eyes were mounted in a perfusion system fitting. Perfusion needles were placed to establish mean baseline pressure. A perfusion pump was connected to the posterior chamber and generated an immediate transient pressure elevation. A phase-sensitive optical coherent tomography system imaged and quantified the TM motion. The peak-to-peak TM displacements (ppTMD) were determined, a tissue relaxation curve derived, and a time constant obtained. This study showed that the ppTMD increased with a rise in the pulse amplitude. The ppTMD was highest for the lowest mean pressure of 16 mmHg and decreased with mean pressure increase. The pulse frequency did not significantly change ppTMD. With a fixed pulse amplitude, an increase in mean pressure significantly reduced the time constant of recoil from maximum distension. Our research platform permitted quantitation of TM motion responses to designed pulse transients. Our findings may improve the interpretation of new TM motion measurements in clinic, aiding in understanding mechanisms and management.

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 Intercompartmental communication between the cerebrospinal and adjacent spaces during intrathecal infusions in an acute ovine in-vivo model

2022 ◽  
Vol 19 (1) ◽  
Author(s):  
Anthony Podgoršak ◽  
Nina Eva Trimmel ◽  
Markus Florian Oertel ◽  
Sara Qvarlander ◽  
Margarete Arras ◽  
...  
Keyword(s):  
Constant Pressure ◽  
Venous Pressure ◽  
Pulse Amplitude ◽  
Estimation Methods ◽  
In Vivo Model ◽  
Mean Pressure ◽  
Quantitative Understanding ◽  
Pressure Changes ◽  
Intrathecal Space

Abstract Introduction The treatment of hydrocephalus has been a topic of intense research ever since the first clinically successful use of a valved cerebrospinal fluid shunt 72 years ago. While ample studies elucidating different phenomena impacting this treatment exist, there are still gaps to be filled. Specifically, how intracranial, intrathecal, arterial, and venous pressures react and communicate with each other simultaneously. Methods An in-vivo sheep trial (n = 6) was conducted to evaluate and quantify the communication existing within the cranio-spinal, arterial, and venous systems (1 kHz sampling frequency). Standardized intrathecal infusion testing was performed using an automated infusion apparatus, including bolus and constant pressure infusions. Bolus infusions entailed six lumbar intrathecal infusions of 2 mL Ringer’s solution. Constant pressure infusions were comprised of six regulated pressure steps of 3.75 mmHg for periods of 7 min each. Mean pressure reactions, pulse amplitude reactions, and outflow resistance were calculated. Results All sheep showed intracranial pressure reactions to acute increases of intrathecal pressure, with four of six sheep showing clear cranio-spinal communication. During bolus infusions, the increases of mean pressure for intrathecal, intracranial, arterial, and venous pressure were 16.6 ± 0.9, 15.4 ± 0.8, 3.9 ± 0.8, and 0.1 ± 0.2 mmHg with corresponding pulse amplitude increases of 2.4 ± 0.3, 1.3 ± 0.3, 1.3 ± 0.3, and 0.2 ± 0.1 mmHg, respectively. During constant pressure infusions, mean increases from baseline were 14.6 ± 3.8, 15.5 ± 4.2, 4.2 ± 8.2, and 3.2 ± 2.4 mmHg with the corresponding pulse amplitude increases of 2.5 ± 3.6, 2.5 ± 3.0, 7.7 ± 4.3, and 0.7 ± 2.0 mmHg for intrathecal, intracranial, arterial, and venous pulse amplitude, respectively. Outflow resistances were calculated as 51.6 ± 7.8 and 77.8 ± 14.5 mmHg/mL/min for the bolus and constant pressure infusion methods, respectively—showing deviations between the two estimation methods. Conclusions Standardized infusion tests with multi-compartmental pressure recordings in sheep have helped capture distinct reactions between the intrathecal, intracranial, arterial, and venous systems. Volumetric pressure changes in the intrathecal space have been shown to propagate to the intraventricular and arterial systems in our sample, and to the venous side in individual cases. These results represent an important step into achieving a more complete quantitative understanding of how an acute rise in intrathecal pressure can propagate and influence other systems.

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 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
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