June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Quantification of Corneal Endothelial Cell Death Due to Compressive Forces
Author Affiliations & Notes
  • Manuel Alejandro Ramirez Garcia
    Biomedical Engineering, University of Rochester, Rochester, NY
  • Yousuf Khalifa
    Emory University, Atlanta, GA
  • Mark Raymond Buckley
    Biomedical Engineering, University of Rochester, Rochester, NY
  • Footnotes
    Commercial Relationships Manuel Ramirez Garcia, None; Yousuf Khalifa, None; Mark Buckley, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1156. doi:
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      Manuel Alejandro Ramirez Garcia, Yousuf Khalifa, Mark Raymond Buckley; Quantification of Corneal Endothelial Cell Death Due to Compressive Forces. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1156.

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

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Abstract
 
Purpose
 

Corneal endothelial cell (CEC) loss from surgical trauma is a critical risk factor for corneal graft failure. However, quantification of the mechanical forces leading to CEC death has yet to be performed. In this study, we developed a testing platform to characterize corneal endothelial cell death as a function of compressive force.

 
Methods
 

Intact corneo-scleral rims were dissected from fresh porcine eyes. Each rim was incubated in a staining solution of Calcein-AM and BSS to visualize endothelial cell viability. Central corneal buttons 8 mm in diameter were punched and immediately placed in a bath of Propidium Iodide (PI) and BSS to asses cell death from the dissecting and trephination procedures. Buttons were submerged for the duration of the experiment. Images were taken before and after loading.<br /> <br /> A custom device utilizing a cantilever beam with a 3 mm stainless steel ball attached was used to indent the endothelium. Experimental groups (Sham, Minimum, Maximum Force, n=3 each) were subjected to peak compressive forces of 0.0 mN, 0.005 mN and 10 mN respectively. Area of cell death was quantified by counting the nuclei of PI-stained cells and multiplying by average cell area.

 
Results
 

No statistical significance was found before and after loading in the sham control or minimum force experimental groups. However, the change in area of cell death in the maximum force experimental group was significant (p = 0.001, Figure 2). Qualitatively, Calcein-AM and PI stained images demonstrated minimal cell loss from dissection and trephination (Figure 1).

 
Conclusions
 

With our current testing platform we are capable of applying compressive forces to the endothelium in the micro and millinewton range in contact areas comparable to surgical tools currently used. The absence of cell death in the sham and minimum force groups indicates we can apply small forces that yield little to no cell death. The presence of significant CEC death in the maximal force group reveals that a critical threshold force exists within the force range tested. Identification of this threshold force may be useful to optimize surgical methods and increase corneal transplant graft survival rates by minimizing endothelial cell loss through mechanical trauma.  

 
Representative specimen before and after indentation for the maximal force (10 mN) group. Calcein-AM (green) stains live cells while PI (red) stains dead cells.
 
Representative specimen before and after indentation for the maximal force (10 mN) group. Calcein-AM (green) stains live cells while PI (red) stains dead cells.
 
 
Error bars represent SEM
 
Error bars represent SEM

 
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