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The Corneal Micropocket Assay: A Model of Angiogenesis in the Mouse Eye
Published on: August 17, 2014
An improved method for examining the corneal endothelium during graft rejection in the rat
F C Figueiredo1, D G Pendergrast, L Zhang
1Department of Ophthalmology, Royal Victoria Infirmary, Newcastle upon Tyne, U.K.
Insights
A new method efficiently isolates rat corneal endothelium for studying immune rejection. This technique reveals that while MHC class I expression remains low, MHC class II and ICAM-1 are significantly upregulated during rejection.
Area of Science:
- Ophthalmology
- Immunology
- Transplantation Biology
Background:
- Studying corneal endothelial pathology requires intact endothelial sheets.
- Assessing immune responses in corneal grafts involves analyzing major histocompatibility complex (MHC) and intercellular adhesion molecule (ICAM)-1 expression.
Purpose of the Study:
- To describe an improved method for obtaining rat corneal endothelial sheets for pathological study.
- To validate the method by examining immunological rejection in corneal transplants.
Main Methods:
- Rat corneal transplants and isografts were performed.
- Corneal stroma was injected with dispase or PBS, followed by cornea removal and fixation.
- Endothelium was peeled, flattened, and stained for morphological and molecular analysis (MHC class I, II, ICAM-1).
Main Results:
- Near-complete endothelial sheets were obtained with minimal background staining.
- MHC class I expression was low and not significantly increased during rejection.
- MHC class II and ICAM-1 were induced de novo and significantly upregulated in allografts compared to isografts.
Conclusions:
- The described method facilitates clear identification of immune mediators in corneal endothelium.
- It confirms low MHC class I expression during rejection.
- It demonstrates de novo induction and strong expression of MHC class II and ICAM-1 during corneal graft rejection.
Abstract:
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.
