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F. Bock, P. Steven, J. Onderka, B. Regenfuß, G. Hüttmann, C. Cursiefen; Three-Dimensional Imaging of Lymphatic Vessels in the Inflamed Cornea by ex-vivo Two-Photon Microscopy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4957. doi: https://doi.org/.
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Due to several diseases and in the course of corneal transplantation the physiologically avascular cornea is invaded by blood and especially lymphatic vessels. To date, detecting and analyzing lymphatic vessel invasion into the cornea is based on ex-vivo fluorescence microscopy that possesses limits regarding spatial resolution. The aim of this study was to analyze the three-dimensional network of inflammatory lymphatic vessels in the cornea by high resolution autofluorescence two-photon microscopy.
In a mouse model of suture-induced inflammatory corneal neovascularization three interrupted 11-0 nylon sutures were placed into the corneal stroma of BALB/c mice (6 weeks old) and left in place for seven days to induce neovascularization. After seven days, mice were sacrificed, corneal whole mounts were prepared and stained with LYVE-1 as a specific lymphatic vascular endothelial marker. The LYVE-1 antibody was detected by a secondary ALEXA-488 conjugated antibody. The three dimensional localization of specific stained lymphatic vessels was performed by standard fluorescence microscopy, followed by two-photon microscopy, that allowed the simultaneous detection of emission spectra of tissue autofluorescence and the labled antibody.
Lymphatic vessels of the limbal arcades were specifically imaged and three dimensionally reconstructed by two-photon microscopy. The simultaneous detection of tissue autofluorescence and ALEXA-488 LYVE-1 antibodies allowed localizing the lymphatic vessel network in relation to the circumjacent tissue at the junction between the epithelium of conjunctiva and cornea. Invading lymphatic vessels sprouted into the corneal stroma and were primarily located directly below the basement membrane extending towards the central cornea.
For the first time, we were able to visualize the three-dimensional network of limbal and corneal lymphatic vessels in the murine cornea by ex-vivo two-photon microscopy. A detailed three-dimensional analysis of the lymphatic vessels system in relation to the circumjacent tissue including epithelium and stromal collagen was demonstrated. The sub-epithelial position of lymphatic vessels suggests a good access for topically applied antilymphangiogenic drugs.
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