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Maria J. Lopez, Takefumi Yamaguchi, Deshea L Harris, Arsia Jamali, Pedram Hamrah; Multi-Photon Intravital Microscopy Of Corneal Draining Lymph Nodes Demonstrates Immune Cell Alterations And Kinetics After Corneal Allotransplantation. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5044.
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To develop a Multi-photon Intravital Microscopy (MP-IVM) model of the corneal draining lymph nodes (dLNs) and to study the spatiotemporal characteristics of dendritic cells (DCs) and T cells, in steady state and after corneal transplantation. To date, studies of these dLN have only been possible in ex vivo and in vitro studies.
MP-IVM (Ultima Pro; Prairie Technologies) was used to image the submandibular dLNs. Anesthetized transgenic mice expressing yellow (YFP) or green fluorescent protein (GFP) for CD11c+ DCs or red fluorescent protein for T cells (Tred) were placed in a custom-build stage and the submandibular dLN was exposed without damaging the vasculature. MP-IVM of the dLNs were performed on mice with normal corneas, as well as for corneas with isograft or allograft transplantation at 7, 14 and 21 days after surgery, during a period of at least 30 minutes. 4D imaging software (Imaris by Bitplane) was used to create 3D movies and analyze for DC kinetics.
MP-IVM imaging of dLNs demonstrated that DCs were distributed predominantly in the parafollicular cortex (T cell zone), but also in the subcapsular area, and sparsely in the follicles (B cell zone) in steady state as well after transplantation. While no significant increase in total DC density (p>0.05) and T cells was noted in isografts, allografts demonstrated a significant increase in DC and T cell densities after 14 and 21 days compared to steady state (p<0.05, 0.01 respectively) and isografts (p=0.03). Further, both isografts and allografts demonstrated increased velocity and track length of donor-derived DC in dLN as compared to controls (p<0.001). However, the meandering index, an index for directionality, which significantly increased in corneal isografts (p<0.001) compared to normal controls, further increased significantly in allografts (p<0.001).
MP-IVM allows visualization of in vivo spatiotemporal kinetics and functionality of DCs and T cells in the murine submandibular dLN. There is an increase in T cell density, DC density and motility after corneal allotransplantation, with increased directionality in allografts. MP-IVM will permit in vivo studies of the behavior of DCs and other immune cells in dLNs, providing an additional window into the corneal immune reflex arc and can be a powerful tool to study the pahogenesis of ocular diseases.
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