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Takefumi Yamaguchi, Kai Hu, Deshea Harris, Pedram Hamrah; Intravital Multiphoton Microscopy of Corneas and Draining Lymph Nodes Shows Increased Velocity of Dendritic Cells after Corneal Transplantation and Directionality in Corneal Allografts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1288.
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The role of dendritic antigen presenting cells in corneal transplantation has been firmly established. While their functional characterization has thus far mainly relied on the analysis of ex vivo studies, there remains a clear need to investigate their behavior in the context of intact tissues in real-time. Here we evaluate in vivo kinetics of dendritic cell (DC) in the cornea and submandibular draining lymph nodes (dLN) after corneal transplantation and trigeminal axotomy as a control for nerve damage model.
Corneal buttons from BALB/c (allogeneic) and C57BL/6 (syngeneic) mice were orthotopically grafted onto CD11c-GFP-DTR (C57BL/6 background) recipients. CD11c-GFP DCs were imaged in the graft, peripheral recipient cornea and dLN under anesthesia using multiphoton microscopy 2 weeks after corneal transplantation and trigeminal axotomy. The density, kinetics and speed of DCs were calculated and 3D movies rendered using high performance 4D imaging software (IMARIS).
The density of CD11c-GFP+ cells in grafts and recipient corneas significantly increased after corneal transplantation and trigeminal axotomy compared to controls (p< 0.01). CD11c-GFP+ cells velocity in grafts significantly increased from 0.58 μm/min (normal, central cornea) and 1.26 (axotomy) to 1.30 (syngeneic), and 1.80 (allogeneic, P<0.001). Velocity of CD11c-GFP+ cells in peripheral recipient cornea significantly increased from 0.94 μm/min (normal, peripheral cornea) and 1.22 (axotomy) to 2.31 (syngeneic), and 2.36 (allogeneic, P<0.001). Velocity of CD11c-GFP+ cells in dLN significantly increased from 2.01μm/min to 2.92 after allogeneic corneal transplantation (P<0.001). The meandering index, an index for directionality, significantly increasedin corneal allografts (p<0.01) compared to isografts or axotomy.
These studies are the first to demonstrate long-term migratory kinetics of corneal DCs after corneal transplantation in both the cornea and submandibular dLNs through high-resolution intravital multi-photon microscopy. DCs demonstrate increased velocity and directionality in the cornea and dLN after allografts compared to axotomy and isografts. Multiphoton microscopy can potentially be a powerful tool to study the pathogenesis of ocular diseases in real-time.
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