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Manuel Alejandro Ramirez Garcia, Yousuf Khalifa, Mark Buckley; Impact of Actin Architecture on the Mechanical Vulnerability of Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1473. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Our previous research has demonstrated that the density of injured corneal endothelial cells (EC) after application of a 9 mN compressive force to the corneal endothelium decreases with increasing death to preservation times (DPT). This study explores the relationship between EC cytoskeletal actin structures to cell loss patterns across DPT.
8 mm-diameter porcine corneal buttons (N = 8 total) were dissected at 0 < t < 12 hrs, 12 < t < 24 hrs, and 24 < t < 48 hrs after slaughter. The endothelium was fixed in 1% PFA for 10 min and then permeabilized and labeled for F-actin in BSS containing 1% DMSO, 5% Triton-X 100 and 165 nM fluorescin-phalloidin for 1.5 hrs at room temperature; specimens were then washed 3x for 30 minutes in BSS. Confocal z-stacks spanning the full EC thickness were taken at 5 distinct locations per fixed specimen.Image analysis was performed in MATLAB. To quantify actin structure, we defined the actin distribution parameter (ADP) as the standard deviation of pixel intensity within a region of interest (ROI). A higher ADP indicates the presence of high contrast features including stress fibers. The ROI was defined as the inner 50% of the cellular area. Cell area was calculated based on cell shape, determined by a manual trace of the cortical actin band in the apical region. Quantification was performed on all cells in all 5 locations averaged per specimen (avg N = 193 cells/specimen). One-way ANOVA was used for statistical analysis.
At 0 < t < 12 hrs, ECs exhibited a cortical double-banded actin structure as well as stress fibers spanning the entire cell (Fig. 1, Fig. 2A). At increasing DPT, the double band disappeared and actin distribution became increasingly homogeneous and diffuse throughout the cytoplasm. As a result, ADP significantly decreased (p=0.007 for 0 < t < 12 hrs vs. 24 < t < 48 hrs) and was less variable at higher DPT (Fig. 1, Fig. 2A).
The presence of stress fibers in ECs at early DPTs could lead to stress concentrations at junctional complexes and increased cell loss when mechanical forces are applied. This effect could explain the previously observed decrease in cell loss with DPT (Fig. 2B). Further research will be targeted exploring this working hypothesis.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
Z-stack slice of the apical cortical actin band of ECs across DPT
A) Quantification of actin features in the inner cytoplasm of ECs. B) Injured cell density at two DPT time intervals
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