Dynamics of Bone Marrow Cells in Normal Corneas and Corneal Allografts of Mice

Abstract

Abstract: : Purpose: The purpose of this study was to evaluate the dynamics of bone marrow cells in normal corneas and corneal allografts using syngeneic bone marrow transplantation model with enhanced green fluorescence protein (EGFP) positive bone marrow cells in mice. Methods: Adult male C57BL/6 mice received 11 Gy of irradiation with a lead eye protector, and were injected with 1 X 10⁷ bone marrow cells prepared from male enhanced green fluorescence protein (EGFP) transgenic mice (C57BL/6 background) through tail vein. Six weeks after GFP⁺ syngeneic bone marrow cell transplantation, some of these C57BL/6 recipients received orthotopic corneal allografts using normal corneas of BALB/c donors, others received no surgery. Normal corneas, corneal allografts and bone marrow were harvested at 2, 4, 8, 12 and 24 weeks after bone marrow transplantation. Bone marrow reconstruction with GFP⁺ bone marrow cells was confirmed by flow cytometry. Whole mounted corneas were stained for cell surface molecules and were observed by confocal microscopy to evaluate the phenotypes and distribution of GFP⁺ bone marrow cells in each period. Results: Approximately 50% of bone marrow cells expressed GFP after bone marrow reconstruction. Confocal microscopic study revealed that in normal corneas a small number of GFP⁺ cells were present only in stroma at early period, and gradually found in all three cellular layers– i.e epithelium, stroma, and endothelium– increasing in number until 12 weeks. Thereafter, the density of these cells was almost constant. These GFP⁺ cells showed no evidence of activated phenotype throughout 24 weeks. On the other hands, GFP⁺ cells in corneal allografts were found in all three layers of epithelium, stroma and endothelium from early period after allo–transplantation. The highest density of these cells was seen at 2 weeks. Thereafter, these cells decreased in numbers until 24 weeks, although a lot of CD11c⁺ and CD11b⁺cells expressing activated phenotypes– i.e CD40, CD80 and CD86– were still found in all three layers. Conclusions: Long time follow up of bone marrow cells in the cornea using syngeneic GFP⁺ bone marrow transplantation model demonstrates that immature bone marrow cells migrate into all three layers of normal cornea and keep constant distribution. After corneal allo–transplantation, bone marrow cells migrate into the allografts with a peek at 2 weeks and then gradually decreases, although activated antigen presenting cells were retained for long time.

Keywords: antigen presentation/processing • inflammation • transplantation