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Sindhu Saraswathy, Goichi Akiyama, Ernesto Barron, Alex Huang; Lymphatic Drainage of Subconjunctival Blebs in Porcine Eyes. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5557. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Previous work has shown that tracers exogenously introduced into the subconjunctival space to create a bleb leads to irregular and linear outflow extensions. Long considered to be lymphatic (given known presence of conjunctival lymphatics), molecular and structural characteristics of just these specific outflow pathways need to be performed.
Tracers (fluorescein, indocyanine green [ICG], or fixable/fluorescent dextrans [500kDa]) were injected into the subconjunctival space of porcine eyes to create a bleb. The blebs and outflow pathways were imaged angiographically or using anterior-segment optical coherence tomography (OCT; Spectralis HRA+OCT; Heidelberg Engineering). The number of outflow pathways were counted in various locations on the eye. In cases using dextrans, 4% PFA was used to trap the tracer to nearby tissues. Conjunctiva was removed with additional angiographic and structural imaging. Dissected conjunctiva were frozen in Tissue-Tek, sectioned (8µm), and imaged (Keyence, BZX700 digital imaging microscope, Chicago, IL, USA). Immunofluroescence was simultaneously performed with a primary antibody against a lymphatic marker (anti goat PROX-1; 1:100 dilution, R&D systems).
In 24 blebs (n = 6 eyes), 16.6/45.8/37.5% showed #0/1/2 outflow pathways respectively. There was no predilection to a particular quadrant. OCT of outflow pathways showed lumens with a clear connection to the bleb internally. OCT of outflow pathways showed clear valves in the direction of fluid flow (Figure.1). Removal of the conjunctiva isolated the fluorescent outflow pathways demonstrating its conjunctival origin. In cases using fluorescent dextrans, fixation specifically trapped the tracer to the lumen allowing identification on histological section. Immunofluorescence with PROX-1 antibody co-localized with the dextrans lining the outflow lumen emanating from the bleb.
Lymphatic outflow from blebs has been long-suspected. In a multi-modal approach, we confirm prior suspicion that lymphatics drain fluid from the subconjunctival space based on the presence of lymphatic-like valves and molecular identification with a lymphatic marker. Future support of lymphatic presence in blebs maybe useful to improve glaucoma surgery outcomes.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
Figure.1: OCT image of the lymphatic vessel with valves in the conjunctiva of Pig.
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