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3D reconstruction of corneal endothelial cell shape: new insight in structure‐function relationships
Author(s) -
HE Z,
BERNARD A,
DEFOE D,
PISELLI S,
ACQUART S,
FOREST F,
DUMOLLARD JM,
PEOC'H M,
GAIN P,
THURET G
Publication year - 2014
Publication title -
acta ophthalmologica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.534
H-Index - 87
eISSN - 1755-3768
pISSN - 1755-375X
DOI - 10.1111/j.1755-3768.2014.4427.x
Subject(s) - membrane , apical membrane , chemistry , cornea , microbiology and biotechnology , magnification , anatomy , biophysics , confocal , biology , optics , computer science , biochemistry , artificial intelligence , physics , neuroscience
Purpose Corneal endothelial cells (CECs) support the pump‐leak theory explaining the maintenance of corneal transparency. CEC are highly polarized with a pole facing aqueous humor having an hexagonal shape bordered by apical tight‐junctions that control permeability. The interdigitated shape of basolateral membranes, described in the late 70’s, are much less known although they bear most of the pumping sites. Aim: to revisit the shape of CEC with modern microscopy and 3D reconstruction. Methods Optimized methods of immunolocalization (fixation and antigen retrieval) were applied on flat mounted whole cornea (fresh or after organ culture). The subcellular structures were distinguished by visualizing apical pole with actin, myosin and ZO‐1; lateral membranes with NCAM (CD56), connexion 43, vimentin and tubulin, and the basal pole with integrin. Stacks of high magnification images were acquired with a confocal microscope with a 150nm pitch. 3D reconstruction was performed with imageJ using convolve and Gaussian blur filters, and a model was defined. Results A 3D map of all markers was obtained. In particular, CD56 perfectly outlined the basolateral membranes of CEC. The 3D model highlighted the dramatic increase in membrane surface due to basolateral expansions. Conclusion CD56 staining pattern and especially the presence of numerous interdigitations should be considered as a hallmark for functional CEC and their study should therefore be integrated in quality controls of CEC production for regenerative medicine processes. Modifications of this 3D structure should also be studied in endothelial diseases. Grant: IFRESIS, St‐ETIENNE METROPOLE