Cellular and Molecular Mechanisms of Corneal Inflammation and Wound Healing

Purpose: Accidental or surgical-induced trauma (e.g., refractive surgery) to the corneal epithelium results in keratocyte death immediately below the wound. Subsequent recovery of keratocytes during healing often remains incomplete, even after many years. Keratocytes produce essential proteins that help maintain corneal stability and preserve corneal clarity, and incomplete keratocyte repopulation after injury can result in loss of normal corneal structure (e.g., keratectasia) and impaired vision. To date, very little is known about the mechanisms regulating keratocyte repopulation. In the mouse, central corneal epithelial abrasion not only evokes keratocyte death, but also neutrophil (PMN), platelet, and γδ T cell recruitment out of the limbal vessels and into the stroma. This inflammatory cell recruitment is necessary for efficient epithelial cell and epithelial nerve recovery. CD18, ICAM-1, and P-selectin are adhesion molecules linked to the regulated recruitment of these inflammatory cells. The purpose of this Dissertation is to determine if the molecular mechanisms regulating inflammatory cell recruitment into the injured cornea are necessary for keratocyte repopulation following central epithelial abrasion. Methods: A 2 mm diameter central epithelial region was mechanically debrided from corneas of male wild type C57Bl/6 (WT), CD18 mutant (hypomorphic), ICAM-1-/-, TCRδ-/- (deficient in γδ T cells) and P-selectin-/-mice. Injured and uninjured corneas were prepared for transmission electron microscopy or immunofluorescence microscopy. PMN-keratocyte interactions, total PMN infiltration (per mm2), total and extravascular (EV) platelet accumulation (per mm2), and keratocyte repopulation were morphometrically analyzed using stereological methods. In vitro analyses, using cultured mouse keratocytes and extravasated PMNs, were performed to investigate PMN motility on keratocytes. Results: Previously, it was determined that PMN CD18 mediated close surface contacts between the PMN and keratocyte. The current studies show keratocyte ICAM-1, a ligand for CD18, also mediates PMN-keratocyte surface contact, and contacts were significantly reduced in ICAM-1-/- mice compared to WT (21.5 ± 3.0% versus 39.9 ± 3.5%, respectively), consistent with the idea that ICAM-1 binds to PMN CD18 to mediate the cell-cell close surface contact. Antibody blockade of PMN CD18 or keratocyte ICAM-1 markedly reduced PMN motility on keratocytes, in vitro (by 33% and 47.5%, respectively), suggesting CD18 and ICAM-1 play a functional role in promoting PMN migration on keratocytes. PMN and platelet recruitment were greatest in ICAM-1-/- mice and, 4 days after corneal abrasion, anterior central (AC) keratocyte numbers returned to baseline, demonstrating ICAM-1 negatively regulates PMN infiltration and platelet accumulation. AC keratocyte repopulation in WT and CD18 mutant mice was significantly lower than their respective baseline counts (by 28% and 56%, respectively). There were no differences in PMN infiltration between WT and CD18 mutant mice but platelet accumulation was blunted in CD18 mutant mice, suggesting platelets, not PMNs, participate in keratocyte recovery. Previous studies show platelet P-selectin and γδ T cells are required for efficient epithelial healing. AC keratocyte repopulation in P-selectin-/- and TCRδ-/- mice only recovered to 31% and 23% of their baselines, respectively. However, infusion of WT platelets into P-selectin-/- mice “rescued” keratocyte repopulation, bringing it back to P-selectin baseline values. Additionally, AC keratocyte repopulation in platelet-depleted WT mice recovered to only 29% of WT baseline. Conclusion: Collectively, these data confirm platelet recruitment is necessary for efficient keratocyte recovery. Interestingly, EV platelet recruitment in TCRδ-/- mice was greater than WT (by 62%) although keratocyte repopulation was low; however, EV platelets in TCRδ-/- mice showed less evidence of shape change, suggesting they were less“activated” in the absence of γδ T cells. The evidence provided in this Dissertation demonstrates a role for adhesion molecules and inflammatory cells in mediating PMN-keratocyte interactions, facilitating PMN migration, regulating inflammatory cell recruitment, and promoting keratocyte repopulation following corneal epithelial abrasion. Collectively, the data suggest this inflammatory cascade is necessary for keratocyte recovery during wound healing.Optometry, College o