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M. I. Melhorn, S. Nakao, A. Hafezi-Moghadam; Depletion of Lyve-1(+) Cells in the Conjunctiva by Clodronate-liposomes. Invest. Ophthalmol. Vis. Sci. 2009;50(13):5519.
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The role of lymphatics in the eye is not well understood. LYVE-1 is a lymphatic marker and is also expressed on certain macrophage subpopulations in the normal eye. Clodronate-liposome (Cl2MDP-LIP) injection is an established method for macrophage depletion. However, whether this method depletes also LYVE-1+cells in conjunctiva is unknown. Here we investigate the effect of Cl2MDP-LIP injection into the subconjunctival space on LYVE-1+and CD11b+ cell numbers.
We generated Cl2MDP-LIP or PBS-liposomes (PBS-LIP) and injected 1-1.5 µL of the liposome stock solution subconjunctivally into mouse eyes. 48 h after injection, eyes were harvested and fixed for 10 min in 4% PFA. Corneal flatmounts were prepared and immunohistochemistry stainings for LYVE-1 and CD11b were performed. Micrographs were obtained and analyzed using ImageJ software.
Injection of Cl2MDP-LIP or PBS-LIP into conjunctiva caused a significant reduction in LYVE-1+ cells outside of the vessels compared to untreated control (5.5±2.6, 14±2.1, 120±36; P=0.004 or P=0.006; n=6). Cl2MDP-LIP significantly reduced LYVE-1+ cell numbers, compared to PBS-LIP (P= 0.03).In limbal area and cornea, PBS-LIP or Cl2MDP-LIP caused a significant increase in CD11b+ cells compared to untreated control (540±65, 571±106, 161±24, P=0.02, n=6).In conjunctiva a significant increase of CD11b+ cells was observed for Cl2MDP-LIP and PBS-LIP, compared to untreated control (P=0.05 for Cl2MBP-LIP and P=0.01 for PBS-LIP; cell numbers: 172±61, 267±66, 10±5.3; n=6), while Cl2MDP-LIP compared to PBS-LIP were not significant (P=0.3; n=6).
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