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S. M. Fleiszig, I. Alarcon, C. Tam, J. J. Mun; Factors that Impact Corneal Epithelial Resistance to Bacterial Penetration and Infection. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5516.
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Factors that determine whether or not P. aeruginosa penetrates the corneal epithelium to cause disease within the stroma are not well understood. The aim of this study was to begin to address that gap in the literature. The approach taken was based on our published data showing that surfactant protein D (SP-D) inhibits bacterial invasion/penetration of corneal epithelium in vitro, and an assumed role for tight junctions in barrier function against infection.
SP-D knockout C57BL/6 mice were compared to wild-type in vivo for susceptibility to epithelial penetration by bacteria. Right eyes were first blotted with tissue paper (Kimwipes) to disrupt superficial epithelial barrier function (to fluorescein) before inoculation with GFP expressing P. aeruginosa strain PA01 (109 cfu in 5 µl). After 5 h, eyes were enucleated and bacterial penetration examined using fluorescence microscopy. Other animals were sacrificed and eyes enucleated immediately after in vivo blotting, and were then pretreated ex vivo for 1 h with 100 mM EGTA (a Ca2+ chelator that disrupts specific cellular junctions). Eyeballs were then placed in 1011 cfu/ml bacteria for 5 h. In other experiments the effects of blotting, SP-D knockout and/or EGTA treatment on disease progression (keratitis) was explored. A 2 mm punched out piece of a soft contact lens was placed on the eye to retain EGTA at the ocular surface for 3 h before challenging with bacteria. Mice were monitored daily for evidence of disease.
The corneal epithelium was found to remain several cells layers thick after blotting. However, blotted eyes stained with fluorescein and showed increased susceptibility to bacterial attachment. Blotting alone did not make corneas susceptible to bacterial penetration beyond the superficial epithelium, nor did it produce susceptibility to keratitis. In contrast, blotting of SP-D knockout eyes, or EGTA treatment after blotting did enable deeper layer bacterial penetration. Accordingly, blotting followed by EGTA treatment promoted susceptibility to disease (visible within 24 hours) that was more severe in SP-D knockouts than in wild-type animals.
The data show that superficial disruption of corneal epithelia can enable fluorescein staining without increasing susceptibility to infection, and that bacteria that bind to superficially disrupted corneas do not necessarily penetrate efficiently into the stroma. They also suggest roles for SP-D and junctional complexes in the mechanism for that resistance to bacterial penetration and ultimately infection.
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