In vitro Generation of Chemotactic Factors Following Keratorefractive Surgery Techniques in Rabbit Corneas

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Abstract
Barraquer keratorefractive cryo techniques currently utilize frozen and lyophilized corneal tissue. Using a Boyden chemotactic assay, we studied the effect of freezing and lyophilization of rabbit corneas on the in vitro release of chemotactic factors for both polymor-phonuclear leukocytes (PMNs) and keratocytes. Treated and untreated corneal buttons (8 mm) were incubated in organ culture (two corneas/ml) for 24 hours at 37C in a 5% CO2 in air incubator. The incubating media was then collected and tested for chemotactic activity using homologous PMNs and keratocytes. Conditioned media with either frozen or lyophilized corneas resulted in significant PMN and keratocyte migration when compared to untreated corneas. When the epithelium and endothelium was removed from the corneas prior to treatment, the level of migratory response decreased but remained significantly above that of untreated corneas. Using a Zigmond-Hirsch checkerboard analysis, the migration activity was demonstrated to be in response to a chemotactic factor rather than to a chemokinetic agent. Heat inactivation of media derived from lyophilized corneas resulted in a decrease in the level of migration activity for both PMNs (45%) and keratocytes (30%). Heat inactivation of media derived from corneal stroma abolished the chemotactic activity for PMNs but resulted in only a 20% decrease in the migration activity for keratocytes. These results indicate that freezing and lyophilization of corneas, as occurs for keratorefractive surgery, results in the generation from the stroma of chemotactic factors for both PMNs and keratocytes. The possible sources of PMN and keratocyte chemotactic factor generated from injured corneas are discussed. The source of the keratocyte chemotactic factor remains unknown but may include fibronectin and collagen fragments.

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