Modulation of Retinal Endothelial Barrier in an in vitro Model of the Retinal Microvasculature

• Published in: Experimental eye research 1996-08, Vol.63 (2), p.211-222
• Main Authors: HASELTON, FREDERICK R., DWORSKA, ELIZABETH, EVANS, STEPHEN S., HOFFMAN, LOREN H., ALEXANDER, STEVEN J.
• Format: Article
• Language: English
• Subjects: Animals; Biological and medical sciences; Blood-Retinal Barrier - drug effects; blood–retinal barrier; Capillaries; Capillary Permeability - drug effects; Cattle; Cells, Cultured; Cytochalasin D - pharmacology; diabetic retinopathy; Endothelium, Vascular - cytology; Endothelium, Vascular - drug effects; Endothelium, Vascular - ultrastructure; Fluorescein; Fluoresceins - pharmacokinetics; Fundamental and applied biological sciences. Psychology; General aspects. Models. Methods; Indicator Dilution Techniques; Isoproterenol - pharmacology; Microspheres; Models, Biological; Molecular Weight; retinal cell culture; retinal microvascular permeability; Retinal Vessels - cytology; Retinal Vessels - drug effects; sodium fluorescein permeability; Vertebrates: nervous system and sense organs; Vitamin B 12 - pharmacokinetics
• ISSN: 0014-4835; 1096-0007
• DOI: 10.1006/exer.1996.0110

The prediabetic/diabetic condition functionally alters the microvascular bed of the eye and the breakdown in the transvascular barrier may be produced by changes in the retinal endothelial barrier. To better understand how retinal microvessel barrier is maintained and is altered in vivo this study applies and extends our previously described in vitro permeability technique to study retinal endothelial monolayers. The model of the retinal microvasculature consists of retinal capillary endothelial cells cultured on porous microcarrier beads and perfused in chromatographic ‘cell-columns’. This model design relies on indicator-dilution techniques to measure the permeability of the retinal endothelial monolayer and detects small changes in retinal endothelial permeability produced by treatments. Bovine retinal capillary endothelial cells (RCE) were obtained using an endothelial selective media. RCE were seeded at 3×10 4cells cm -2of fibronectin-coated gelatin microcarriers. After 7 days of microcarrier culture, microcarriers were poured to form columns 0.66cm in diameter and 1.6cm in length. The cell-column elution patterns of coinjected optically absorbing tracers (blue dextran 2×10 6Da; cyanocobalamin 1355Da; sodium fluorescein 376Da) were analysed to estimate the permeability of the RCE monolayers covering the microcarriers. Scanning electron microscopic examination showed complete monolayer formation on the surface of the microcarriers. We found that baseline monolayer permeability averaged 7.57±0.57×10 -5cmsec -1for cyanocobalamin and 9.29±0.78×10 -5cmsec -1for sodium fluorescein (mean± S.E.M., N=39). Permeability did not increase over 2hr of cell-column perfusion. Permeability was decreased by 1μ m isoproterenol ( n=3) and increased by 1μ gml -1cytochalasin D ( n=5). This is one of the first reports of in vitro permeability values for the transport barrier formed by retinal microvascular endothelial cells. Furthermore, the endothelial component of the retinal barrier is dynamic, and is enhanced by isoproterenol and diminished by cytochalasin D.