Abstract
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.
Highlights
The trabecular meshwork represents a location of great interest for glaucoma research, and non-invasive methods are needed to decellularize it and convert it into a scaffold for cellular transplantation
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
Summary
The trabecular meshwork represents a location of great interest for glaucoma research, and non-invasive methods are needed to decellularize it and convert it into a scaffold for cellular transplantation. In contrast to ab interno trabeculectomy, freeze-thaw decellularization is a nonspecific and site-agnostic method to remove all living cells from eye cultures to ready them for the transplantation and study of cells of interest[14,15]. 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 large change in the distal outflow pattern
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