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J. M. Gonzalez, Jr., M. C. Edman, S. F. Hamm-Alvarez, J. C. H. Tan; Cellular and Matrix Elements in the Aqueous Outflow Pathway of Human Corneoscleral Transplant Tissue. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5776.
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© ARVO (1962-2015); The Authors (2016-present)
To examine features of cells, such as nuclei and actin cytoskeleton, and structural extracellular matrix in situ in the aqueous outflow tissue of human corneoscleral rims used for transplantation.
Trabecular meshwork tissue was explanted from human corneoscleral transplant rims that were up to one week post-mortem. The outflow tissue was teased out as a strip from corneoscleral rims, cut into smaller pieces and placed on glass microscope slides. The tissue was fixed with 4% paraformaldehyde for 30 minutes, washed, permeablized with Triton-X 100 0.5% for 2 min, then blocked with 3% BSA for 2 h at 4°C. It was then stained with 1:50 phalloidin-rhodamine for 30 minutes, washed in PBS, then mounted flat in media containing DAPI. The specimens were then examined by laser confocal and multiphoton microscopy.
DAPI-stained nuclei were scattered throughout the thickness of the trabecular meshwork. Deep in the tissue the distribution of nuclei seemed to take the form of a monolayer. Nuclear fragmentation was not observed. Filamentous actin staining (568nm) could be seen throughout the thickness of the explanted outflow tissue. Cortical-like F-actin distribution was evident especially deep in the tissue. A dense network of autofluorescent fibers that were prominent by 488nm light stimulation traversed the whole tissue, although this was sparser in its deepest layer. The imaged fibers were consistent with a vast elastin network, as corroborated by multiphoton second harmonic imaging at 750nm.
We describe an approach for visualizing cell nuclei, filamentous actin cytoskeleton and elastin networks in the outflow tissue of transplant corneoscleral rims. The findings suggest the more superficial parts of the explanted tissue as examined represent the lamellated meshwork while its deeper parts were consistent with the juxtacannalicular meshwork-Schlemm's canal inner wall endothelial complex.
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