Evaluation of an in vitro Model of Neutrophil Migration in the Equine Cornea

Abstract

Abstract: : Purpose: The purpose of this study is to develop an in vitro model of neutrophil migration in equine cornea. Once characterized, this model can be used to evaluate methods to block migration and prevent neutrophil related pathology in the cornea. Methods: Fresh equine corneas were collected from normal eyes of horses euthanized for reasons unrelated to ocular disease. The corneas were mounted in a dialysis cell assay that had 1cm diameter reservoirs on both the epithelial and endothelial surfaces of the cornea. One milliliter of purified equine neutrophils (5x10⁶/ml) were added to the epithelial chamber. Serial dilutions of leukotriene B4 (LTB4) in HBSS⁺⁺ (1uM, 100nM, 10nM) were added to the endothelial chamber as a neutrophil chemoattractant to determine a dose response relationship. HBSS⁺⁺without LTB4 was used for a control. The assays were then incubated for 2 hours at 37C. The fluid from both chambers was collected and neutrophil counts were performed. This experiment was then repeated with neutrophils pretreated with a beta-2 integrin specific adhesion receptor antibody (50 ug IB4) and a control anti-equine MHC class 1 antibody (50 ug B5C). Results: Cell counts performed on fluid from each chamber showed a dose dependent response in neutrophil migration across the cornea. Higher concentrations of LTB4 induced migration of larger numbers of neutrophils across the cornea to the endothelial chamber (68 x 10⁴, 54.5 x 10⁴, 24 x 10⁴ and 6 x 10⁴ at 1uM, 100nM, 10nM and control, respectively). In addition, pretreatment of the neutrophils with IB4 blocked migration to numbers similar to the control cornea (6 x 10⁴). Pretreatment with B5C had minimal effect. Conclusions: An in vitro whole mount corneal migration assay may be useful for evaluating treatments designed to block neutrophil migration into the cornea. The data from this study suggests a role for beta-2 integrins in neutrophil adhesion and migration in the equine cornea. Although further study is needed, this data also suggests that medications or antibodies that block integrin activation or function may be useful treatment options in keratitis.

Keywords: keratitis • cornea: basic science • animal model