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OBJECTIVE Previous studies from this laboratory have demonstrated, in a rabbit model of keratitis, a relationship between the corneal virulence of Staphylococcus aureus and the alpha-toxin activity of the infecting bacteria. This study is a histopathological characterization of the action of purified alpha-toxin on corneal tissue. METHODS Alpha-toxin was purified by isoelectric focusing and intrastromally injected into rabbit corneas (2 micrograms per cornea). A kinetic analysis of toxin effect was performed following injection. Normal corneas and corneas injected with phosphate buffered saline (PBS) or heat-inactivated alpha-toxin in PBS served as controls. Eyes were examined from 0 to 4 h by slit lamp examination (SLE) and scored on the basis of seven ocular parameters. Corneal tissue was removed and examined for histopathological changes. RESULTS From 0.5 to 4 h post-injection, alpha-toxin injection induced a significant increase in the SLE score relative to untreated eyes or eyes injected with PBS (P < 0.0001). Histolo-pathological examination of corneas one-half h after alpha-toxin injection revealed edema of the central cornea and death of epithelial cells by both necrosis and apoptosis. Later times showed continued edema and loss of apparently normal epithelial cells. Development of polymorphonuclear (PMN) leukocyte infiltration from the tear film into the central cornea and from limbal vessels into the peripheral cornea was observed. CONCLUSIONS Purified alpha-toxin mediates cell death by necrosis and apoptosis, sloughing of viable corneal epithelial cells, severe corneal edema, and PMN migration into the cornea from both the tear film and limbal vessels. The pathologic changes revealed by histological studies of corneas injected with purified alpha-toxin included death of cells by necrosis and apoptosis as well as overall changes analogous to that seen by SLE of eyes infected with wild-type, but not alpha-toxin-deficient strains of Staphylococcus aureus.
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