Human Tear Fluid Protects Mouse Cornea against Pseudomonas aeruginosa Keratitis

Abstract

Abstract: : Purpose: We previously showed that human tear fluid protects corneal epithelial cells against P. aeruginosa in vitro by blocking bacterial invasion and by reducing toxin mediated cell injury. The aim of this study was to determine how human tear fluid effects the development of corneal infection in vivo. Methods: The effect of human tear fluid on bacterial colonization and virulence was evaluated using a mouse scratch model. Reflex tear fluid was collected from the conjunctival sac of human volunteers using a microcapillary tube. Corneas were challenged with various concentrations (10³, 10⁴, 10⁵, or 10⁶ CFU) of P. aeruginosa strain 6206 prepared either in full concentration human tear fluid, or in cell culture media (MEM control). One group of mice was inoculated immediately after scratching the left cornea, while a second group was inoculated after scratched corneas were allowed to heal for 6 hours. Severity of disease was graded in a masked fashion after 24 and 48 h. Animals were then sacrificed and the corneas were individually homogenized in sterile tissue grinders for enumeration of viable bacteria. Other animals were sacrificed 1 or 4 h after inoculation to determine the effect of tears on early colonization by bacteria. To interpret the results of colonization experiments, the effect of tears on bacterial adherence to corneal cells in vitro was examined using cells grown on 0.4 µm filters. Results: Human tear fluid was found to reduce the incidence and the severity of disease caused by P. aeruginosa (p<0.05), with greatest effects on the 6 h heal model and with inocula of 10⁴ CFU or less. The mechanism was found to involve reduced bacterial colonization detectable 1 h after inoculation (median 4.2 x 10³ vs 350), which increased in magnitude by 4 h (8.5x10³ vs 40). Interestingly, tear fluid had little effect on bacterial adherence to cells in vitro (1.3+0.6x10⁵ vs 0.9±0.2x10⁵, p=0.4 ). Conclusions: The in vivo results showed tear fluid protection against infection involving reduced colonization early in the infectious process. The in vitro data suggested that the mechanism may not involve reduced adherence to corneal epithelial cells. Whether or not in vitro and in vivo protection against P. aeruginosa induced damage involves the same tear components is yet to be determined.

Keywords: keratitis • cornea: tears/tear film/dry eye • Pseudomonas