Lee Ellen J, Evans David J, Fleiszig Suzanne M J
Morton D. Sarver Laboratory for Cornea and Contact Lens Research, School of Optometry, University of California at Berkeley, Berkeley, CA 94720-2020, USA.
Invest Ophthalmol Vis Sci. 2003 Dec;44(12):5220-7. doi: 10.1167/iovs.03-0229.
The scarified cornea keratitis model was modified to study Pseudomonas aeruginosa infection of healing corneal epithelium. The new model was then used to study the role of ExsA, a transcriptional activator of P. aeruginosa, in bacterial penetration through injured and healing corneal epithelia.
Scratch-injured corneas of C57BL/6 mice were allowed to heal for 0, 6, 9, or 12 hours before inoculation with a cytotoxic (6206) or invasive (PAO1) P. aeruginosa strain. Disease progression was monitored for 14 days. The integrity of the healing epithelium was studied in uninfected eyes by fluorescein staining and by histologic examination. In other experiments, the effect of bacterial exsA mutation was studied after 0, 6, or 12 hours of healing. Three hours after infection, these eyes were used to quantify early bacterial colonization levels by viable counts, or they were sectioned to study bacterial penetration through the epithelium by microscopy.
Corneas remained susceptible to infection 6 but not 12 hours after scratch injury. By 6 hours, the previously exposed stroma was already completely covered by several layers of epithelial cells. Fluorescein staining unexpectedly occurred even after 12 hours of healing time, showing that resistance to infection preceded full restoration of epithelial barrier function. Mutation of exsA reduced both bacterial colonization levels and penetration through the epithelium 3 hours after bacterial inoculation, but only in the 6-hour healing situation, and only for the cytotoxic strain (PA103). Mutation of exsA in the invasive strain (PAO1) had no effect on 3-hour colonization or penetration levels under any circumstances.
The 6-hour healing infection model showed a role for ExsA in early interactions with the corneal epithelium that was not detectable with the conventional (0-hour) scratch model. Comparison of the 6- and 12-hour healing models, which showed that factors additional to barrier function contribute to defense against infection, could be used to gain new insights into corneal defense mechanisms, and the methods used by bacteria to circumvent them.
对划痕角膜角膜炎模型进行改良，以研究铜绿假单胞菌对愈合角膜上皮的感染情况。然后利用该新模型研究铜绿假单胞菌转录激活因子ExsA在细菌穿透受损及愈合角膜上皮过程中的作用。
C57BL/6小鼠角膜划痕损伤后，在接种细胞毒性（6206）或侵袭性（PAO1）铜绿假单胞菌菌株前分别让其愈合0、6、9或12小时。监测疾病进展14天。通过荧光素染色和组织学检查研究未感染眼愈合上皮的完整性。在其他实验中，研究愈合0、6或12小时后细菌exsA突变的影响。感染3小时后，这些眼睛用于通过活菌计数量化早期细菌定植水平，或者进行切片以通过显微镜研究细菌穿透上皮的情况。
角膜在划痕损伤后6小时仍易受感染，但12小时后则不然。到6小时时，先前暴露的基质已被几层上皮细胞完全覆盖。即使在愈合12小时后仍意外出现荧光素染色，表明对感染的抵抗力先于上皮屏障功能的完全恢复。exsA突变在细菌接种3小时后降低了细菌定植水平以及穿透上皮的能力，但仅在6小时愈合情况下如此，且仅针对细胞毒性菌株（PA103）。侵袭性菌株（PAO1）中的exsA突变在任何情况下对3小时的定植或穿透水平均无影响。
6小时愈合感染模型显示ExsA在与角膜上皮的早期相互作用中发挥作用，这在传统的（0小时）划痕模型中无法检测到。6小时和12小时愈合模型的比较表明，除屏障功能外的其他因素有助于抵抗感染，可用于深入了解角膜防御机制以及细菌规避这些机制的方法。
