Abstract
Purpose :
To assess changes in the elasticity of ex vivo human optic nerve tissue due to exogenous addition of various cholesterol species, including esterified cholesterol, using atomic force microscopy (AFM).
Methods :
Optic nerve tissue (total n=25) was procured from human cadaveric donor eyes (with equal gender distribution, donor age average 70.5 ± 6.5 year, all Caucasian donors) with no known history of ocular diseases. Optic nerves were transversely sectioned, and these sections were secured to the bottom of the petri dish using a topical “liquid bandage” formulation (CVS Health); the tissue was then immersed in PBS to maintain hydration. Young’s Modulus was determined using a custom designed AFM through microindentation (2mm diameter spherical tip, 0.1N/m spring constant). Force-indentation curves were acquired from measurements and analyzed using custom MATLAB code. Young’s modulus of elasticity was determined using the Hertz model for a spherical indenter. Measurements were repeated up to 15 times at 3 different points. For each sample, elasticity was assessed prior to and immediately after a 15-minute treatment period. Tissue sections were treated with 5 µM lipids (Cholesterol Ester (20:3), Cholesterol, Zymosterol, and 15-alpha-hydroxycholestane), or phosphate buffered saline (PBS) as a control for 15 minutes. The results were normalized using the average elastic modulus values from each experiment and a two-tailed t-test was performed.
Results :
All samples had a significantly different (p<0.05, n=5) Young’s modulus of elasticity pre- and post-treatment with exogenous lipids (Cholesterol Ester (20:3), Cholesterol, Zymosterol, and 15-alpha-hydroxycholestane). There was no significant difference for the samples treated with PBS control. There was an increase in Young’s modulus (increase in stiffness) for cholesterol (154%), Zymosterol (190%), 15-alpha-hydroxycholestane (30%) and a decrease in Young’s modulus (decrease in stiffness) for Cholesterol Ester (56%).
Conclusions :
Our results suggest that cholesterol and its precursors such as Zymosterol and 15-alpha-hydroxycholestane increase the stiffness of the optic nerve, whereas unsaturated Cholesterol Ester (20:3) decreases its stiffness.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.