Abstract
Abstract: :
Purpose: To determine if limited exposures to cytochalasin B can inititate mitosis in the organ cultured corneal endothelium and if this cell proliferation is dependent on MAPK signaling. Methods: Corneas with a surrounding ring of sclera were isolated from Sprague-Dawley rats and placed in basal media Eagle (BME) containing 10% rabbit serum (RS), 50µg/ml of insulin and 0.25µg/ml of cytochalasin B (CB) for times ranging from one to six hours. After CB exposure, tissues were transferred into BME containing 10% RS and insulin (50µg/ml). After 24 h colchicine was added to the media to collect mitotic figures at a final concentration of 10-8M. After a total culture time of 48 h, tissues were fixed, flat mounted, and observed. In another series of experiments, media was supplemented with 25-100 mM of PD98059, an inhibitor of MAPK. After 1 h in the media, endothelia were removed and given circular freeze injuries. They were then placed back into the media for the duration of the experiment. At 24 h colchicine was added and the tissues fixed 24 h thereafter. PD98059 treatment (up to 100 mM) was also used in conjunction with CB treatment to determine if MAPK inhibition would adversely effect endothelial mitosis stimulated by exposure to the alkaloid. Results: Limited CB exposure for up to 6 h produced a substantial mitotic response in the organ cultured rat endothelium. This response diminished with shorter CB exposure times. Furthermore, the presence of PD98059 (up to 100 mM) in the media did not inhibit this mitotic response. Likewise, the mitotic response in injured tissues was also unaffected by PD98059 at the concentrations stated above. In all cases, when tissues were examined 48 h after wounding they all displayed prominent mitotic responses around the wound area. Conclusions: These results indicate that a 6 h limited exposure to CB can initiate a substantial mitotic response in the non-injured organ cultured rat corneal endothelium. The mitotic response, whether induced by a limited exposure to CB or freeze injury, can occur in the presence of the MAPK inhibitor PD98059. This suggests that MAPK may not play a critical role in endothelial cell proliferation.
Keywords: cornea: endothelium • proliferation • signal transduction