Vision Science Program, University of California, Berkeley, CA, United States of America.
School of Optometry, University of California, Berkeley, CA, United States of America.
PLoS One. 2020 May 29;15(5):e0234013. doi: 10.1371/journal.pone.0234013. eCollection 2020.
The healthy cornea is remarkably resistant to infection, quickly clearing deliberately inoculated bacteria such as Pseudomonas aeruginosa and Staphylococcus aureus. Contrasting with the adjacent conjunctiva and other body surfaces, it also lacks a resident viable bacterial microbiome. Corneal resistance to microbes depends on intrinsic defenses involving tear fluid and the corneal epithelium. Dry eye, an ocular surface disease associated with discomfort and inflammation, can alter tear fluid composition and volume, and impact epithelial integrity. We previously showed that experimentally-induced dry eye (EDE) in mice does not increase corneal susceptibility to P. aeruginosa infection. Here, we explored if EDE alters corneal resistance to bacterial colonization. EDE was established in mice using scopolamine injections and dehumidified air-flow, and verified by phenol-red thread testing after 5 and 10 days. As expected, EDE corneas showed increased fluorescein staining versus controls consistent with compromised epithelial barrier function. Confocal imaging using mT/mG knock-in mice with red-fluorescent membranes revealed no other obvious morphological differences between EDE corneas and controls for epithelium, stroma, and endothelium. EDE corneas were imaged ex vivo and compared to controls after alkyne-functionalized D-alanine labeling of metabolically-active colonizing bacteria, or by FISH using a universal 16S rRNA gene probe. Both methods revealed very few viable bacteria on EDE corneas after 5 or 10 days (median of 0, upper quartile of ≤ 1 bacteria per field of view for each group [9-12 eyes per group]) similar to control corneas. Furthermore, there was no obvious difference in abundance of conjunctival bacteria, which included previously reported filamentous forms. Thus, despite reduced tear flow and apparent compromise to corneal barrier function (fluorescein staining), EDE murine corneas continue to resist bacterial colonization and maintain the absence of a resident viable bacterial microbiome.
健康的角膜对感染具有很强的抵抗力，能够迅速清除故意接种的细菌，如铜绿假单胞菌和金黄色葡萄球菌。与相邻的结膜和其他体表面不同，角膜也缺乏常驻的有活力的细菌微生物组。角膜对微生物的抵抗力取决于涉及泪液和角膜上皮的内在防御。干眼症是一种与不适和炎症相关的眼表面疾病，它可以改变泪液成分和体积，并影响上皮完整性。我们之前曾表明，在小鼠中诱导的实验性干眼症 (EDE) 不会增加角膜对铜绿假单胞菌感染的易感性。在这里，我们探讨了 EDE 是否会改变角膜对细菌定植的抵抗力。通过注射东莨菪碱和除湿空气流在小鼠中建立 EDE，并在第 5 和 10 天用苯酚红线测试进行验证。正如预期的那样，EDE 角膜的荧光素染色比对照增加，这与上皮屏障功能受损一致。使用带有红色荧光膜的 mT/mG 敲入小鼠进行共聚焦成像显示，EDE 角膜与对照相比，上皮、基质和内皮之间没有其他明显的形态差异。将 EDE 角膜进行离体成像，并与代谢活跃定植细菌用炔基功能化 D-丙氨酸标记后的对照进行比较，或用通用 16S rRNA 基因探针进行 FISH。两种方法都显示在 EDE 角膜上 5 或 10 天后存活的细菌很少（每组中位数为 0，每个视野的上限四分位数≤1 个细菌[每组 9-12 只眼睛]），与对照角膜相似。此外，结膜细菌的丰度没有明显差异，其中包括以前报道的丝状形式。因此，尽管泪液流量减少且角膜屏障功能明显受损（荧光素染色），但 EDE 小鼠角膜仍能抵抗细菌定植，并保持缺乏常驻的有活力的细菌微生物组。
