Countermeasures against vesicant-induced epithelial de-adhesion in the cornea

Sulfur mustard is a chemical weapon and vesicant (blister-inducer) that causes severe effects to the cornea. These injuries are characteristically delayed in healing and may be recurrent over time. The experiments in this dissertation were designed to test the efficacy of countermeasures targeting metalloproteinases, thought to be over-activated in the cornea following vesicant exposure. An ex vivo culture model was developed using dissected corneas from young adult rabbit eyes. Each cornea was exposed drop wise to 20 nmol 2-chloroethyl ethyl sulfide (CEES, half mustard) or 100 nmol nitrogen mustard (NM). These are less potent and less toxic analogs of sulfur mustard, and induce mild or moderate injury, respectively. Twenty-four hours after NM exposure there are observable separations between the epithelium and the stroma, termed microbullae. I hypothesize that the microbullae are caused by activation of the enzyme ADAM17 (a disintegrin and a metalloprotease 17), causing cleavage of the transmembranous anchoring protein, collagen XVII. Four ADAM17 inhibitors were compared to evaluate the effect of attenuating activity of this enzyme. The countermeasures effectively improved the appearance of the epithelial-stromal junction as seen by the preservation of epithelial-stromal attachments and improved histology as well as and decreasing ADAM17 activity. After vesicant exposures there was also upregulation of MMP-9, the protease responsible for the necessary matrix degradation after wounding that leads to healing. The MMP-9 levels after vesicant exposure are enhanced abnormally. The MMP inhibitors, doxycycline and minocycline, were employed as effective countermeasures to inhibit the prolonged upregulation of MMP-9. To determine whether activation of ADAM17 and MMP-9 are due to ERK signaling, the inhibitor PD98059 was assessed. When used immediately after exposure, the compound was able to inhibit the ability of ERK to phosphorylate the cytoplasmic domain of ADAM17, thereby inhibiting collagen XVII cleavage. MMP-9 upregulation after 24 hrs was also inhibited as a downstream affecter of the ERK pathway when PD98059 was used. These experiments identify which countermeasures are the best candidates to test in vivo in rabbits exposed to sulfur mustard, and explore a mechanism of how mustards affect the extracellular matrix of the epithelial-stromal junction.Ph. D.Includes bibliographical referencesIncludes vitaby Andrea DeSantis Rodrigue