Fig 4. RTX treatment reduces bacterial burden in the infected eyes.

A. Schematic representation of the experimental approach. Subcutaneous inoculation of RTX was given to four-week-old mice. The mice were treated for 3 consecutive days, with incrementing dosages; day one: 30 μg/kg; day two: 70 μg/kg; day three: 100 μg/kg. Mice were rested and infected at 6 weeks of age with 5x10⁵ CFU/ml of P. aeruginosa 6294. Ocular tissues were harvested for analysis. B. Representative images of sham (left) and infected (right) eyes at 24h post-challenge. Data are representative from three independent experiments with N = 6 mice per treatment per experiment. C. Corneal sensitivity was captured by measuring the blinking thresholds of mice longitudinally, commencing with RTX treatment and ending with infection. The RTX treatment caused a mild, but significant decrease of blinking reflexes (asterisk, p = 0.003, Two-way ANOVA) that was observed only a week post-treatment, followed by complete recovery. The blinking threshold significantly declined in infected mice under both treatments. Data are representative from three independent experiments with N = 6 mice per treatment per experiment. D. Representative images of stained for β-tubulin shams and infected corneal whole mounts from RTX- and vehicle-treated mice, scale bar = 20 μm. The z-stack covers 40 μm from corneal epithelium to stroma with 1 μm interval and is shown as 2D projection. The images were analyzed using custom script in Fiji to quantify stained areas per image. Six whole-mounts per group per treatment were analyzed and values plotted (right panel; p-values by One-way ANOVA; ns, not significant). The quantification of β-tubulin-stained areas shows decrease of β-tubulin in cornea periphery upon RTX treatment and with infection. E. Mouse scratch-injured corneas were infected with 5x10⁵ CFU/ml of P. aeruginosa 6294 and corneal and conjunctival tissues were harvested at 24h post-challenge. Bacterial burdens were determined by plate counts (Student’s t-test, p = 0.0001). Corneal pathology was depicted by box and whiskers plots showing 25% and 75% ranges (Mann-Whitney comparison, p = 0.01). Data are representative from three independent experiments, each containing cohorts with N = 6 mice per treatment per experiment. Cumulatively, data show that the reduction in neuronal fibers abundance consequent to RTX treatment did not cause sustained alterations in blink reflexes. Significant changes in pain sensation were observed only consequent to infection irrespective of RTX treatment. RTX treatment resulted in decreased P. aeruginosa burden in the infected corneas.