Effects of the synthetic cannabinoid WIN 55,212–2 on corneal epithelial wound healing in vitro

Abstract

Abstract: : Purpose:Topical application of cannabinoids has been explored for glaucoma treatment. Previous reports have shown that the use of eye drops is associated with ocular irritation. The corneal epithelium has CB₁ receptors, although their function in this tissue is unknown. The purpose of this study was to evaluate the effect of the synthetic CB₁ agonist WIN 55,212–2 on corneal epithelial wound healing. Methods:Transformed human corneal epithelial cells were cultured to a confluent monolayer. A scratch wound was performed, and the cells were exposed to different concentrations of the synthetic CB₁ agonist WIN 55,212–2. The effect of a simultaneous addition of the CB₁ antagonist AM251, as well as the incubation of the cell cultures with this antagonist were also explored. For control purposes, the cultures were kept in medium alone, or were added dimethyl sulphoxide (DMSO), the solvent used for the CB₁ agonist and antagonist. The wound area was video–monitored in a microscope stage incubator at constant 37°C, and the images were stored in an image analyser. Results:The wound area of the untreated cultures healed completely after 22.0±4.583 hours, the one treated with DMSO after 29.0±3.12 hours. WIN 55,212–2 prevented the wound healing, ultimately leading to cell death. Incubation of the cell cultures with AM251 10–12 hours before the addition of WIN 55,212–2 (1 nMol/ml) resulted in complete wound closure. However, when AM 251 and WIN 55,212–2 were added simultaneously to the cell monolayer, wound healing was prevented. Conclusions:WIN 55,212–2 prevented corneal epithelial wound healing in a human cell line of corneal epithelium in vitro, leading to cell death. Incubation of the cell cultures with the antagonist AM251 prior to treatment with the synthetic CB₁ agonist WIN 55,212–2 allowed the normal epithelial wound healing to occur, suggesting the possibility of a CB–receptor mediated effect. This findings may have relevance for the possible future therapeutic application of topical cannabinoids in glaucoma.

Keywords: cornea: epithelium • wound healing • clinical laboratory testing