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M.I. Roche, A. Hsia, L. Van Parijs, V.L. Perez; Real Time Imaging of Bone Marrow-Derived Inflammatory Cell Migration into the Cornea During Lipopolysaccharide Induced Keratitis . Invest. Ophthalmol. Vis. Sci. 2003;44(13):702.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To establish an in vivo technique to visualize in real time the migration of bone marrow derived inflammatory cells into the cornea during the induction of keratitis. Methods: GFP bone marrow chimera mice were generated to trace the migration of inflammatory cells into the cornea. Bone marrow-derived stem cells were first harvested from the tibia and fibula of mice treated with 5-FU. After expansion in vitro, bone marrow stem cells were infected with a retroviral vector expressing GFP. GFP positive transfected cells were then transplanted into a lethally irradiated mouse and reconstitution was confirmed by flow cytometry. Keratitis was induced by intrastromal injection of LPS (2ug) in the cornea of GFP chimera mice. Migration pattern of GFP cells into the cornea was evaluated at different time points by in vivo real time imaging, using fluorescent microscopy and digital camera with time lapse software. Immunohistochemical analysis was done ex-vivo to identify infiltrating GFP positive inflammatory cells. Results: GFP positive cells were detected in the peripheral blood, thymus, spleen and bone marrow of GFP bone marrow chimera mice by flow cytometry. Bone marrow-derived GFP cells were present in the corneal limbus prior to treatment. In vivo microscopy showed migration of GFP positive cells from the limbus into the cornea as early as 30 minutes after intrastromal LPS injection. Time lapse analysis revealed a dynamic and random pattern of GFP positive cells migrating from limbus to limbus across the cornea. Immunohistochemical staining of corneal specimens demonstrated the presence of neutrophils and macrophages in the cornea. Conclusion: Real time visualization of inflammatory cells migration into the cornea can be accomplished in GFP chimera mice using time lapse in vivo imaging. Bone marrow-derived inflammatory cells reside in the limbus and rapidly migrate across the cornea in response to LPS. We believe that real time imaging in the cornea is a novel technique that will enhance the understanding of mechanisms involved in the recruitment of neutrophils and macrophages into sites of inflammation
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