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I. Alarcon, L. Kwan, C. Yu, S.M. J. Fleiszig; The Role of Epithelial Basement Membrane in Corneal Defense Against P. Aeruginosa . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1912.
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Among the roles attributed to epithelial basement membranes is the ability to polarize epithelium by anchoring cells and regulating solute trafficking, wound healing, tissue regeneration and repair. Previously we showed that invasive P. aeruginosa can cross multilayered corneal epithelium in vitro; yet bacteria do not penetrate into underlying stroma of uninjured corneas in vivo. We hypothesized that the basement membrane of the corneal epithelium, a meshwork containing pores (range 0.022–0.216 µm) smaller than bacteria (1.0 µm), might modulate bacterial penetration.
Rabbit corneal epithelium was grown in vitro on 8µm pore–sized filters with or without artificial basement membrane (Matrigel, pore size 26–359). Cells were inoculated apically with 106 CFU of invasive P. aeruginosa. Every hour for 5 h, viable counts were taken from the upper and lower chambers, and transepithelial resistance (TER) recorded. GFP–labeled bacteria were used for fluorescence microscopy. Mouse corneas were used (uninjured, superficially injured with filter paper, or scratch injured to the anterior stroma) to explore bacterial penetration and subsequent disease outcome.
Matrigel hindered bacterial passage through corneal epithelium for both high (PAO1) and low (PAK and PAO1 lasA/lasB/AprA mutant) protease producing strains of P. aeruginosa. Matrigel did not increase TER, yet microscopy 1 h post–infection showed relative retention of bacteria at the superficial surface of cells grown on Matrigel. By 3 h, significant numbers of bacteria penetrated into epithelium grown with and without Matrigel. At 5 h, bacteria accumulated along the surface of filters under cells only when grown on Matrigel, suggesting Matrigel mediated trapping. Accordingly, Matrigel without cells reduced bacterial penetration through filters. In vivo and in organ culture, bacteria entered the stroma only where injury breached the epithelial cell basement membrane. Otherwise and elsewhere, bacteria penetrating the corneal epithelium accumulated along the underlying basement membrane.
These results showed in vitro that artificial basement membrane can modify bacterial penetration through corneal epithelium. The mechanism was found to involve increased resistance to bacterial penetration at the superficial cell surface, in addition to a direct physical barrier effect at the other side of the cell layer. These controlled in vitro cell culture findings corresponded with descriptive studies done using whole corneas to suggest that the basement membrane is a defense against infection.
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