An improved method for examining the corneal endothelium during graft rejection in the rat

An improved method of removing rat corneal endothelial sheets for study of endothelial pathology is described. The method was validated by examining morphological changes and changes in expression of major histocompatibility complex (MHC) and intercellular adhesion molecule (ICAM)-1 on endothelium undergoing immunological rejection. PVG strain rats received LEW strain corneal transplants or corneal isografts. Just prior to and during graft rejection, animals were killed, together with a group of untreated animals. The corneal stroma was injected with dispase or PBS, the cornea was carefully removed, fixed in acetone and the endothelium was gently peeled off and flattened on to a glass slide. Morphological changes, together with MHC class I, class II and ICAM-1 expression were visualised by immuno-histochemical staining and quantified by image analysis. Near complete endothelial sheets were obtained by this method. Because of the thin cell layer, there was minimal background staining, permitting rejection-associated changes to be clearly seen. MHC class I expression on normal endothelium was low and not significantly increased on endothelial cells of allografts at the time of rejection compared with controls (P = 0.1). MHC class II and ICAM-1 were induced de novo, expression being significantly higher on allografts than on isografts (P = 0.004 for MHC class II and P = 0.01 for ICAM-1). MHC class I and II and ICAM-1 were expressed on many infiltrating cells. Thus, this preparation method permits clear identification of the distribution and morphology of infiltrating cells and other mediators of the immune response in the entire donor endothelium. It confirms that MHC class I expression is low during rejection, while MHC class II and ICAM-I are induced de novo and strongly expressed.