The Effect of Combined Shear Stresses on Epithelial Cell Killing by P. aeruginosa

Abstract

Abstract: : Purpose:Using an in vitro model system, we have shown that shear stress reduces corneal epithelial cell killing by cytotoxic P. aeruginosa (Lakkis et al ARVO 2001). Single shear stresses within the range expected during daily hydrogel lens wear, with pause times correlating with normal blink intervals, were found to reduce but not totally eliminate cytotoxicity. As combinations of shear stresses are exerted on the ocular surface during blinking, our aim was to test the hypothesis that combined shear stresses will completely eliminate P. aeruginosa cytotoxicity. Methods:A rocker apparatus was used to generate combinations of shear stresses over the surface of rabbit corneal epithelial cells (10₆ cells/mL) grown on tissue culture treated coverslips. Angular velocity was varied to create a range of shear stresses over the surface of the cells, which were infected with 10₆ CFU/mL of the cytotoxic P. aeruginosa strain 6206. The first angular velocity of the rocking motion was set to be twice that of the second, as might be expected to occur during blinking. These cells were compared to uninfected control cells (media alone). Periods of zero shear stress (pause times between rocking), representative of normal blink intervals, were also incorporated. Trypan blue dye exclusion assays were used to quantify cytotoxicity. Results:A decline in cytotoxicity of corneal epithelial cells was observed with the application of a range of combined shear stresses (100.58/50.29 - 134.11/67.05 dynes/cm₂, 5 s pause), when compared to stationary conditions. No combination of shear stresses completely prevented cell damage. When combinations of shear stresses were increased in an attempt to totally eliminate cytotoxicity, epithelial cells were damaged by the shear stresses before cytotoxicity was completely prevented. Higher combined shear stresses could be applied with increased pause times; however, protection from cytotoxicity decreased as pause time increased. Conclusion:A combination of shear stresses, as might be expected to occur during blinking, appears to reduce but not completely eliminate cytotoxicity. In vivo, factors other than shear stress might contribute to protecting the cornea from P. aeruginosa cytotoxicity.

Keywords: 531 Pseudomonas • 372 cornea: epithelium • 367 contact lens