Age-Related Differences in the Mouse Corneal Epithelial Transcriptome and Their Impact on Corneal Wound Healing.

Purpose: Aging is a risk factor for dry eye. We sought to identify changes in the aged mouse corneal epithelial transcriptome and determine how age affects corneal sensitivity, re-epithelialization, and barrier reformation after corneal debridement. Methods: Corneal epithelium of female C57BL/6J (B6) mice of different ages (2, 12, 18, and 24 months) was collected, RNA extracted, and bulk RNA sequencing performed. Cornea sensitivity was measured with an esthesiometer in 2- to 3-month-old, 12- to 13-month-old, 18- to 19-month-old, and 22- to 25-month-old female and male mice. The 2-month-old and 18-month-old female and male mice underwent unilateral corneal debridement using a blunt blade. Wound size and fluorescein staining were visualized and photographed at different time points, and a re-epithelialization rate curve was calculated. Results: There were 157 differentially expressed genes in aged mice compared with young mice. Several pathways downregulated with age control cell migration, proteoglycan synthesis, and collagen trimerization, assembly, biosynthesis, and degradation. Male mice had decreased corneal sensitivity compared with female mice at 12 and 24 months of age. Aged mice, irrespective of sex, had delayed corneal re-epithelialization in the first 48 hours and worse corneal fluorescein staining intensity at day 14 than young mice. Conclusions: Aged corneal epithelium has an altered transcriptome. Aged mice regardless of sex heal more slowly and displayed more signs of corneal epithelial defects after wounding than young mice. These results indicate that aging significantly alters the corneal epithelium and its ability to coordinate healing.

Ectoine, from a Natural Bacteria Protectant to a New Treatment of Dry Eye Disease.

Ectoine, a novel natural osmoprotectant, protects bacteria living in extreme environments. This study aimed to explore the therapeutic effect of ectoine for dry eye disease. An experimental dry eye model was created in C57BL/6 mice exposed to desiccating stress (DS) with untreated mice as controls (UT). DS mice were dosed topically with 0.5-2.0% of ectoine or a vehicle control. Corneal epithelial defects were detected via corneal smoothness and Oregon Green dextran (OGD) fluorescent staining. Pro-inflammatory cytokines and chemokines were evaluated using RT-qPCR and immunofluorescent staining. Compared with UT mice, corneal epithelial defects were observed as corneal smoothness irregularities and strong punctate OGD fluorescent staining in DS mice with vehicle. Ectoine treatment protected DS mice from corneal damage in a concentration-dependent manner, and ectoine at 1.0 and 2.0% significantly restored the corneal smoothness and reduced OGD staining to near normal levels. Expression of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) and chemokines CCL3 and CXCL11 was significantly elevated in the corneas and conjunctivas of DS mice, whereas 1.0 and 2.0% ectoine suppressed these inflammatory mediators to near normal levels. Our findings demonstrate that ectoine can significantly reduce the hallmark pathologies associated with dry eye and may be a promising candidate for treating human disease.

Conditional deletion of CD25 in the corneal epithelium reveals sex differences in barrier disruption.

PURPOSE: IL-2 promotes activation, clonal expansion, and deletion of T cells. IL-2 signals through its heterotrimeric receptor (IL-2R) consisting of the CD25, CD122 and CD132 chains. CD25 knockout (KO) mice develop Sjögren Syndrome-like disease. This study investigates whether corneal CD25/IL-2 signaling is critical for ocular health. METHODS: Eyes from C57BL/6 mice were collected and prepared for immunostaining or in-situ hybridization. Bulk RNA sequencing was performed on the corneal epithelium from wild-type and CD25KO mice. We generated a conditional corneal-specific deletion of CD25 in the corneal epithelium (CD25Δ/ΔCEpi). Corneal barrier function was evaluated based on the uptake of a fluorescent dye. Mice were subjected to unilateral corneal debridement, followed by epithelial closure over time. RESULTS: In C57BL/6 mice, CD25 mRNA was expressed in ocular tissues. Protein expression of CD25, CD122, and CD132 was confirmed in the corneal epithelium. Delayed corneal re-epithelization was seen in female but not male CD25KO mice. There were 771 differentially expressed genes in the corneal epithelium of CD25KO compared to wild-type mice. While barrier function is disrupted in CD25Δ/ΔCEpi mice, re-epithelialization rates are not delayed. CONCLUSIONS: All three chains of the IL-2R are expressed in the corneal epithelium. Our results indicate for the first time, deleting CD25 systemically in all tissues in the mouse and deleting CD25 locally in just the corneal epithelium compromises corneal epithelial barrier function, leading to dry eye disease in female mice. Future studies are needed to delineate the pathways used by IL-2 signaling to influence cornea homeostasis.