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L. Ma, J. Li, K. Kuang, P. Iserovich, J. Fischbarg; Further Evidence for Paracellular Fluid Transport by Corneal Endothelium . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2909.
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We have priorly generated evidence suggesting that that the corneal endothelium transports fluid through the paracellular route. For this we have used fluorescent particles as fluid flow markers, and FITC–dextran 70 kD as a lateral intercellular space marker that piles up against the apical ends of the spaces. Given our initial fluorescence microscopy chamber geometry, we have used primary cultures of bovine corneal endothelial cells grown on coated Anopore filters.
To buttress the initial results using more adequate cultured cell supports, and animal preparations.
(a) We developed methodology to mount in our chamber cultured cells grown on 12 mm Transwell cup inserts. For this, supports carrying cells were glued to the chamber (with inert silicone elastomer), then cut from the cup as desired. (b) In addition, we modified Dikstein–Maurice chambers so that rabbit corneas can be observed in vitro with an inverted microscope. (c) Lastly, we also used whole rabbit eyes held in a chamber for microscopic observation of the cornea and anterior chamber. In all experiments, corneal endothelial cells were visualized by loading them for 30 min with calcein AM and using 484/517 nm for excitation and emission, respectively. For the fluorescent particles we used 555/605 nm. Respective images could be later overlaid. This allowed us to focus at all times on a plane at the same distance to the cell surface (6 µm).
Particle trajectories, acceleration at fixed spots, distributions of particle velocities, and pile up of extracellular marker are in good qualitative agreement with those seen with the Anopore supports.
The data add support to the hypothesis of endothelial fluid transport by the paracellular route.
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