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A. L. Moyer, M. Harrington, M. C. Callegan; Bacillus cereus Induces Disruption of Tight Junctions of an in vitro Blood Retinal Barrier. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4729.
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During Bacillus endophthalmitis, immune cells infiltrate into the eye, which may be a result of a breach in the blood retinal barrier. Therefore, we developed an in vitro model of the blood retinal barrier and evaluated permeability and tight junction alterations of the retinal pigment epithelium (RPE) upon infection with Bacillus cereus.
RPE were cultivated on collagen-, fibronectin-, and protein-coated glass coverslips in RPE monolayer media. DAPI-stained single-cell layers were visualized by confocal microscopy. Polarization of these mononlayers was confirmed by examining expression and localization of the Na, K-ATPase and tight junction proteins by immunoblot and immunocytochemistry. Polarized RPE monolayers were infected with 0.5 ml of 102 CFU of B. cereus and examined at 0, 2, 4, 6, and 8 hours postinfection. Changes in RPE monolayer permeability were analyzed by transepithelial resistance measurements and a dextran conjugate permeability assay. Tight junction proteins were examined by immunoblot and immunocytochemistry. Cell cytotoxicity was accessed by trypan blue exclusion assay and a lactate dehydrogenase release assay.
Light microscope and confocal examination of cells indicated that RPE grown in this fashion formed monolayers which expressed and demonstrated an apical distribution of the Na, K-ATPase and intact occludin and ZO-1. During infection, changes in the barrier function of RPE monolayers paralleled disruptions in tight junction proteins. Bacillus infection of RPE resulted in increased permeability to FITC-dextran and a loss of transepithelial resistance with time. Localization of ZO-1 and occludin at the cell periphery was lost by 4 hours postinfection, and protein expression of both tight junction proteins was lost by 8 hours postinfection. These results were corroborated by maximal cell cytotoxicity by 8 hours postinfection.
RPE can be grown in vitro as polarized monolayers, mimicking the blood retinal barrier. Upon infection with Bacillus, RPE monolayers lost barrier function in conjunction with alterations in tight junction proteins. Future studies will focus on determining the mechanism of tight junction disruption and barrier breakdown, as well as the effect of these events on loss of retinal structure and function during Bacillus endophthalmitis.
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