Abstract
Abstract: :
Purpose: To re–evaluate CD34 antigen expression in normal mouse cornea using dual and triple staining techniques Methods: Wholemount preparations of mouse and rat corneas were examined with confocal microscopy using single, dual or triple immunostaining to study their morphology, phenotype and distribution. Single cell suspensions from normal mouse corneas were also prepared and analysed by flow cytometry. After short term culture of corneal stromal explants nonadherent cells were harvested, cytospins prepared and stained for different markers. Results: Combined staining for F–actin and leukocyte differentiation markers clearly showed that the corneal stroma contains a population of CD45+ resident bone marrow–derived cells, while the major proportion of cells were CD45–F–Actin+ keratocytes. A significant proportion (two thirds) of CD45+ cells in the normal corneal stroma expressed CD34+ while no CD45– cells (ie keratocytes) co–expressed CD34. In addition, CD34+ cells were CD11c–, CD11b+. Less then 10% of the CD34+ cells also co–expressed Sca–1+, but no CD34+ cells co–expressed MHC class II+. In contrast, the remaining population of CD45+ CD34– cells in the corneal stroma expressed CD11b, MHC Class II+ but not CD11c, and were found mostly in anterior and peripheral part of stroma. These cells are in intimate contact with corneal keratocytes which stained only for F–actin and were negative for all leukocyte markers. A very few CD45+ cells expressed the B220 marker suggesting a plasmacytoid dendritic cells phenotype. Flow cytometry analyses confirmed the morphometric data showing that 68% of CD45+ cells co–express CD34 and CD11b while 22% are CD11b+, CD34–.Conclusions: Mouse keratocytes are negative for CD34. The normal mouse cornea contains two populations of bone marrow derived leukocytes, both of which are distinct from stromal keratocytes. The larger population are CD34+ and resemble haemopoietic stem cells while the smaller population are CD34–CD11b+ MHC Class II+ macrophages. A very small percentage comprise plasmacytoid dendritic cells.
Keywords: cornea: stroma and keratocytes