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Danielle M Robertson, Matthew Petroll; Second harmonic generation imaging of corneal stroma after infection by Pseudomonas aeruginosa. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3291.
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© ARVO (1962-2015); The Authors (2016-present)
Second harmonic generation (SHG) imaging allows for the visualization of collagen lamellae orientation in the corneal stroma and has been used to evaluate stromal changes following injury or during wound healing. The purpose of this study was to evaluate the directional movement and orientation of Pseudomonas aeruginosa (PA) in the corneal stroma using SHG imaging.
PA strain 6487, an invasive corneal isolate stably conjugated to GFP, was used in this study. PA was suspended in RPMI media at a concentration of ~10^8 CFU/mL using a spectrophotometer and confirmed by colony counts. Ex vivo rabbit corneas were subject to three partial thickness wounds at a depth of 75 µm using a diamond knife. Corneas were inoculated with PA and incubated at 37°C for 0, 2, 6, 18, 24, and 48 hours. Corneas incubated in RPMI media alone were used as controls. At the indicated time points, corneas were washed with PBS to remove non-adherent PA, fixed in 1% paraformaldehyde and mounted epithelial side down on coverslip bottom dishes. The central, mid-peripheral, peripheral and limbal stroma were imaged using multiphoton fluorescence and SHG imaging. Image stacks were reconstructed 3 dimensionally and analyzed for PA localization and orientation. Orientation with respect to the collagen lamellae was quantified using the Directionality plug-in in ImageJ.
At the initial 2 hour time point, PA localized to the corneal surface in the area of the scratch wound and was present in the wound valley. By 6 hours, early movement of PA into the stroma from the wound margin was visible. At 18 hours, the patterning of PA within the collagen stroma paralleled the orientation of the lamellae. This effect was further enhanced at 24 and 48 hours.
In the infected cornea, PA spreads through the stroma along and parallel to the collagen lamellae. The distinct differences in the structure and organization of collagen lamellae in the central and limbal cornea may contribute to the susceptibility of the paracentral cornea to infection as compared to the limbal region.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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