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B. Bourghardt Peebo, N. Lagali, P. Fagerholm; Indicators of Corneal Lymphatics: Comparision of Immunohistochemistry With in-vivo Confocal Microscopy. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3717.
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To compare lymph vessel morphology in whole-mount corneas stained with LYVE-1 with morphological features of suspected lymphatics observed in a live cornea using in-vivo confocal microscopy.
Corneal angiogenesis was induced in twenty male rats under general anaesthesia by suture placement in one cornea of each animal. 7 to 14 days after suture placement, animals were anaesthetized and corneas were examined with in-vivo confocal microscopy. Corneas were then removed and prepared for immunohistochemistry. Whole-mount corneas were co-stained with LYVE-1 and CD31 for simultaneous visualization of lymph and blood vessels with a fluorescence microscope. The identity of vessels (blood or lymph) in the stained corneas was then compared with the morphology of vessels observed in-vivo.
In stained corneas, lymph vessels were found less frequently than blood vessels. Lymph vessels in the limbus had a wider diameter than blood vessels, and a rough, discontinuous surface with bulging protrusions. Lymph vessels invading the cornea were similar in diameter to corneal blood vessels but with evidence of thinner vessel walls. In-vivo confocal microscopy revealed dark, wide-diameter vessel-like structures in the limbus with thin walls and an absence of erythrocytes. Limbal blood vessels could be clearly distinguished by the presence of erythrocytes. In the cornea, blood vessels could be clearly identified on the basis of erythrocyte flow, while a subpopulation of corneal vessels contained flowing material but without the presence of discrete particles. A further subpopulation of corneal vessels appeared dark or transparent within the vessel walls with no apparent particles or flow characteristics.
In-vivo confocal microscopy of neovascularized rat corneas revealed a small population of vessel-like structures, not present in untouched control eyes, exhibiting distinct morphologic differences from blood vessels. Comparison with stained whole mount corneas indicates that the population of vessels observed in-vivo exhibits morphology consistent with immunohistochemically-confirmed lymphatic vessels.
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