B6 mice were subconjunctivally injected with LiCl in contrast to appropriate control sodium chloride (NaCl), and then routinely infected with PA. Clinical score, slit-lamp photography, hematoxylin and eosin (H&E) staining, and bacterial plate counts were used to determine the role of LiCl in PA keratitis. Messenger ribonucleic acid and protein levels of inflammatory cytokines in PA-challenged mouse corneas and in vitro cultured macrophages and neutrophils were measured with real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively. Apoptosis of the infiltrating inflammatory cells in the PA-infected murine corneas was assessed using terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling staining and propidium iodide staining associated with flow cytometry. In cultured murine macrophages and neutrophils, cell apoptosis was determined with annexin V/propidium iodide double staining associated with flow cytometry and western blot analysis for cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase.
Treatment with LiCl reduced the severity of corneal disease by reducing corneal inflammatory response and bacterial burden. Moreover, LiCl increased anti-inflammatory cytokine interleukin-10 levels, decreased proinflammatory cytokine tumor necrosis factor-α levels, and enhanced apoptosis of infiltrating macrophages and neutrophils in the PA-infected mouse corneas. In vitro studies further confirmed that LiCl elevated anti-inflammatory cytokine expression but reduced proinflammatory cytokine production, as well as promoted cell apoptosis in murine macrophages and neutrophils.