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This ex vivo study aims to describe the morphological characteristics of the human limbal lymphatic vasculature using confocal microscopy and immunofluorescence. Twelve corneal specimens are obtained from human donor eyes. These corneal buttons are not suitable for transplantation due to low endothelial cell count. After the central button of the corneas were removed, each corneoscleral rim were cut into eight pieces, resulting in 96 limbal samples. For laser scanning confocal microscopy, 48 limbal explants were dried on the endothelial side with a cellulose sponge and held fixed in front of the applanation cap. Bowman’s layer were identified first centrally, then moved toward the limbus until Bowman’s layer terminates. En-face images were captured. The rest of the limbal explants were fixed in 4% formaldehyde and mounted with Tissue Tek optical coherence tomography (OCT) compound. Half of these samples were used for immunofluorescene analysis, using Podoplanin and CD-31 as markers for lymphatic and blood vessels respectively; whilst immunohistochemical analysis was performed on the rest of the samples with LYVE1 and CD-31 staining to identify lymphatic and blood vessels. The immuno-analysis sections were compared to confocal microscopy image reconstruction with an in-house automated program written in Matlab. The authors confirmed that in contrast to blood vessels, lymphatic vessels display no discernible vessel wall, are filled sparsely with cells, have a dark lumen and show very slow flow. Based on en-face analysis, the authors also added that limbal blood vessel complex and the lymphatic vessel complex are morphometrically different in segment length and vessel diameters. As lymphageogenesis is the principal mediator of corneal graft rejection, further characterisation of limbal lymphatic vasculature on confocal microscopy could potentially make it a valuable non-invasive imaging modality for monitoring treatment and assessing individual risks. The translational potential of this study is limited by the fact that ex vivo limbal tissues were used for confocal microscopy assessment, so the morphology of the lymphatic vessels may vary from in vivo settings.

En-face analysis of the human limbal lymphatic vasculature.
Palme C, Ahmad S, Romano V, et al.
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Siyin Liu

Manchester Royal Eye Hospital, UK.

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