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Mouse corneal lymphangiogenesis model

Nature Protocols volume 6, pages 817–826 (2011)Cite this article

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Abstract

This protocol describes a powerful in vivo method to quantitatively study the formation of new lymphatic vessels in the avascular cornea without interference of pre-existing lymphatics. Implantation of 100 ng of lymphangiogenic factors such as vascular endothelial growth factor (VEGF)-A, VEGF-C or fibroblast growth factor-2, together with slow-release polymers, into a surgically created micropocket in the mouse cornea elicits a robust lymphangiogenic response. Newly formed lymphatic vessels are detected by immunohistochemical staining of the flattened corneal tissue with lymphatic endothelial-specific markers such as lymphatic vessel endothelial hyaluronan receptor-1; less-specific markers such as vascular endothelial growth factor receptor 3 may also be used. Lymphatic vessel growth in relation to hemangiogenesis can be readily detected starting at day 5 or 6 after pellet implantation and persists for ∼14 d. This protocol offers a unique opportunity to study the mechanisms underlying lymphatic vessel formation, remodeling and function.

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Figure 1: Schematic diagram of pellet preparation, insertion and angiogenic response in the mouse cornea.
Figure 2: Analysis of FGF-2-induced lymphatic vessels by LYVE-1 and VEGFR3 staining and blood vessels by CD31 staining.
Figure 3: Examples of corneal angiogenesis and lymangiogenesis induced by various angiogenic factors.

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Acknowledgements

Our laboratory was supported through research grants from the Swedish Research Council, the Swedish Cancer Foundation, the Karolinska Institute Foundation, the Karolinska Institute distinguished professor award, the European Union Integrated Project of Metoxia (Project no. 222741) and the European Research Council Advanced Grant ANGIOFAT (Project no. 250021). Y.C. is supported by the Swedish Heart and Lung Foundation.

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  1. yihai.cao@ki.se

Authors and Affiliations

  1. Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden

    Renhai Cao, Sharon Lim, Hong Ji, Yin Zhang, Yunlong Yang, Jennifer Honek & Eva-Maria Hedlund

  2. Department of Medicine and Health Sciences, Linköping University, Linköping, Sweden

    Yihai Cao

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  1. Renhai Cao
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Contributions

Y.C. designed the study. R.C. conducted the experiments. R.C., S.L., H.J., Y.Z., Y.Y., J.H., E.-M.H. and Y.C. analyzed the data. R.C., S.L., H.J., Y.Z., Y.Y., J.H. and Y.C. participated in designing and discussing this study. R.C., S.L. and Y.C. wrote the paper.

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Cao, R., Lim, S., Ji, H. et al. Mouse corneal lymphangiogenesis model. Nat Protoc 6, 817–826 (2011). https://doi.org/10.1038/nprot.2011.359

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