The Effect of Hyperglycemia on Gut Microbial diversity, Ocular Surface Inflammation and Corneal Re-epithelialization after Alkaline Chemical Injury in Mice

Abstract

Purpose 1)To compare corneal re-epithelialization rates and ocular surface inflammatory response after corneal alkali burn injury between Akita (diabetic) and wild type mice. 2)To compare gut microbial diversity between diabetic and wild type mice at baseline and 72 hours after alkaline burn injury and to correlate this with ocular surface findings. Methods Alkaline burn injury was induced on the right eye of mice under general anesthesia, with a piece of 1.5mm diameter filter paper soaked in 0.1 – 1M NaOH solution placed on the center of the cornea for 5-30 seconds. For optimization experiments, wild type mice (7-9 weeks) were placed in groups with differing NaOH concentrations and exposure durations (n=5 per group). The time to complete corneal re-epithelialization and tear cytokine levels were compared between Akita and wild type mice (7-9 weeks) on day 0, 3, 7 and10 after injury. Baseline and 72-hours post injury gut microbial diversity was also compared after metagenomic shotgut sequencing between wild type and Akita mice. Results For 1M NaOH concentrations, the percentage of 5, 10 and 30 second-exposure groups achieving complete re-epithelialization by day 10 was 50%, 25% and 0% respectively. For 0.1M NaOH concentrations, the percentage of 10 and 30 second-exposure groups achieving complete re-epithelialization at day 10 was 100% and 80% respectively. From the experiments, 0.1M NaOH with 10 seconds exposure was determined to be the optimal concentration and exposure time for our experiments. Regarding microbial diversity, firmicutes, bacteriodetes and proteobacteria were the predominant bacterial commensals at baseline. Between groups, clostridia and epsilonproteobacteria were significantly more abundant in wild type mice, while bacteroidia, actiinobacteria, gammaproteobacterial and firmicutes were more abdunant in Akita mice at baseline. Conclusions 0.1M NaOH and 10 seconds are the optimal NaOH concentration and exposure time to induce a corneal epithelial defect and ocular surface inflammatory response for our subsequent experiments, as the injured cornea shows gradual recovery and was able to heal within 10 days. With the initial sequencing result of the baseline gut microbime pattern of wild type and Akita mice, we may further explore the difference in gut microbiome after inducing the chemical injury on the two groups.