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R. Vargas-Pinto, K. Perkumas, W. D. Stamer, D. Ho, M. Johnson; Young’s Modulus of Schlemm’s Canal Cells Using Atomic Force Microscopy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2131.
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© ARVO (1962-2015); The Authors (2016-present)
The cells of the inner wall endothelium of Schlemm’s Canal (SC) likely participate in the regulation of the aqueous humor outflow. The stiffness of SC cells determines the magnitude of the transcellular pressure gradient that they can support. We used Atomic Force Microscopy (AFM) with different tip sizes to assess the relative contribution of cell membrane and cytoskeleton in supporting load.
Cultured SC cells were stained with Calcein AM and Hoechst fluorescent dyes to determine cell viability and the location of the nucleus. Force measurements were made on isolated cells using AFM with silicon nitride sharp pyramidal tips (angle between 25 and 45°) and tips with polystyrene spherical attachments (tip diameters of 4.5 and 10 µm). Young’s Modulus (E) was calculated using the modified Hertz model and a nominal angle of 35° for the tip.
Young’s Modulus for SC cells measured with pyramidal tips was found to be 9.7± 4.1 kPa (n=11) in the nuclear region, while outside the nucleus, E was 28.2± 22.9 kPa (n=10). When using tips with spherical attachments, much lower values were obtained both in the nuclear region (4.5 µm tip: 0.58± 0.32 kPa, n=14; 10 µm tip: 0.30± 0.24 kPa, n=8) and outside of the nucleus (4.5 µm tip: 1.41± 0.53 kPa, n=5; 10 µm tip: 0.30± 0.20 kPa, n=3). E obtained using spherical tips were similar to those reported using magnetic bead cytometry (1-3kPa), while the value calculated using pyramidal tips is at least an order of magnitude larger.
The significantly higher modulus of SC cells measured using pyramidal tips suggest that their plasma membranes can play an important role in determining the stiffness of these cells, indicating that it may be important to compare SC cell membrane stiffness in glaucomatous cells with those of normal SC cells.
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