June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Dexamethasone increases the cortical stiffness of Schlemm’s canal endothelial cells
Author Affiliations & Notes
  • Amir Vahabikashi
    Biomedical Engineering, Northwestern University, Evanston, IL
  • Kristin Marie Perkumas
    Ophthalmology, Duke University, Durham, NC
  • William Stamer
    Ophthalmology, Duke University, Durham, NC
    Biomedical Engineering, Duke University, Durham, NC
  • Mark Johnson
    Ophthalmology, Northwestern University, Chicago, IL
    Biomedical and Mechanical Engineering, Northwestern University, Evanston, IL
  • Footnotes
    Commercial Relationships Amir Vahabikashi, None; Kristin Perkumas, None; William Stamer, None; Mark Johnson, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 6133. doi:
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    • Get Citation

      Amir Vahabikashi, Kristin Marie Perkumas, William Stamer, Mark Johnson; Dexamethasone increases the cortical stiffness of Schlemm’s canal endothelial cells. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):6133.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

Reduced outflow facility in glaucoma is thought to be associated with decreased pore formation on the inner wall endothelium of Schlemm’s Canal (SC). Recent evidence (Overby, PNAS, 2014) indicates that elevated mechanical stiffness of glaucomatous SC cells contributes to reduced pore formation in glaucomatous eyes. Corticosteroids are known to affect cell stiffness and can cause ocular hypertension. We here examine the effect of dexamethasone on SC cell stiffness.

 
Methods
 

Normal (n=2) and glaucomatous (n=2) SC cell strains were treated with 0 (Control), 0.01μM, 0.1μM, 1μM of dexamethasone for 7 days. Atomic Force Microscopy (AFM) was then used to characterize cortical and subcortical stiffness using sharp (20 nm) and spherical tips (10 μm), respectively (15-20 cells in each group). Two-tailed t-tests were used for statistical analysis. Samples were then fixed with 4% paraformaldehyde, stained with Alexafluor 568 Phalloidin (F-actin) and Hoechst (nucleus), respectively, and then were imaged using confocal microscopy.

 
Results
 

When compared to controls (Figure 1), cell strains showed 1.5-2.1 fold higher cortical stiffness at a dexamethasone concentration of 1 μM (p<0.05), while differences with lower doses were not significant. No difference was found in subcortical stiffness between treatment doses in any cell strain. Imaging showed increased stress fibers with increasing dexamethasone concentration (Figure 2). No change in cortex thickness was seen. No differences were seen between normal and glaucomatous cell stains.

 
Conclusions
 

Our results suggest that dexamethasone treatment increases cell stiffness, which may impede pore formation in the inner wall endothelium and thereby increase outflow resistance and potentially cause glaucoma. Increased assembly of actomyosin machinery might be the responsible mechanism for elevated cortical stiffness. However, in spite of increased stress fibers caused by dexamethasone treatment, we saw no change in subcortical stiffness. This suggests that other cytoskeletal elements, possibly intermediate filaments, play a more dominant role in regulation of the subcortical stiffness.  

 
Figure 1. AFM results for cortical and subcortical stiffness of normal and glaucomatous SC cells.
 
Figure 1. AFM results for cortical and subcortical stiffness of normal and glaucomatous SC cells.
 
 
Figure 2. SC cells labeled for F-actin (red) and cell nuclei (blue). (A) Control, (B) 0.01μM, (C) 0.1μM, and (D) 1μM. Thick arrows point to the cortex and thin arrows point to stress fibers.
 
Figure 2. SC cells labeled for F-actin (red) and cell nuclei (blue). (A) Control, (B) 0.01μM, (C) 0.1μM, and (D) 1μM. Thick arrows point to the cortex and thin arrows point to stress fibers.

 
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