Alkali burn-induced synthesis of inflammatory eicosanoids in rabbit corneal epithelium

Alkali burning of the rabbit cornea is a well-established model for the study of anterior surface inflammation, neovascularization, and wound-healing processes. 12-hydroxyeicosanoids have been implicated as mediators of such responses. 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE) is a lipoxygenase-derived arachidonate metabolite and 12(R)-hydroxyeicosatetraenoic acid (12[R]-HETE) is formed by a cytochrome P450 monooxygenase; both give rise to the potent angiogenic factor 12(R)-hydroxyeicosatrienoic acid (12[R]-HETrE). In this study, the authors correlate the pattern of their synthesis in the corneal epithelium with the inflammatory response after alkali injury. New Zealand albino rabbits were anesthetized and alkali burns created with 10-mm filter paper discs (1 N NaOH for 2 minutes). Corneas were then rinsed; 1 to 7 days later, corneal epithelium was scraped and used to assess 14C-arachidonic acid conversion to 12-HETE and 12-HETrE enantiomers in the presence of NADPH by chiral high-pressure liquid chromatography. The inflammatory response secondary to the alkali burn was quantified through area measurements of reepithelialization and neovascularization. Alkali burn induced a time-dependent production of corneal epithelial 12-HETE and 12-HETrE. A marked increase in 12-HETE and 12-HETrE synthesis was evident at day 2 (from 22 +/- 7 to 139 +/- 22 ng/hour) after injury, increasing to 800 +/- 68 ng/hour at day 7. Chiral analysis revealed a time-dependent synthesis of the R and S enantiomers of 12-HETE (24% R, 76% S) and 12-HETrE (72% R, 28% S). Total arachidonate metabolism, as well as the formation of 12(R)-HETrE, correlated with the area of neovascularization (P < 0.01 and P < 0.02, respectively). The results demonstrate that surviving and regenerating epithelium has an increased capacity of synthesizing 12(S)-HETE and 12(R)-HETE and that maximal production of 12(R)-HETrE, a known direct and indirect angiogenic factor, coincides with neovascularization in this model. Thus, the lipoxygenase and cytochrome P450-dependent activities increased in a time-dependent manner, indicating the potential involvement of both pathways in the inflammatory response to alkali burn. The formation of significant quantities of 12(R)-HETE and 12(R)-HETrE is a novel finding in this alkali injury model.