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Jorawer S. Singh, Joyce E. Young, Sangita P. Patel; Paracellular Permeability In Corneal Endothelial Monolayers Of Varying Density. Invest. Ophthalmol. Vis. Sci. 2012;53(14):5345.
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The paracellular pathway is a primary route for water movement across the corneal endothelial monolayer. Although fluid influx is known to increase at low monolayer densities, underlying changes in the paracellular pathway are not well defined. This study investigates alterations in transendothelial electrical resistance (TER) and diffusional paracellular permeability (Papp) in corneal endothelial monolayers of varying endothelial cell density (ECD) to test the hypothesis that monolayers of lower ECD have lower TER and greater Papp.
Bovine eyes were obtained from local abattoirs. Passage 1 bovine corneal endothelial cells were seeded onto 0.33 cm2 permeable polycarbonate transwell inserts in two different media at varying dilutions to obtain monolayers of different ECD (DMEM + 10% FBS, 1:4, for ECD 1500-2999 cells/mm2; MEM + 0.5% FBS, 1:16, for ECD 1000-1499 cells/mm2). TER was monitored using an epithelial voltohm meter in an Endohm chamber (World Precision Instruments, Sarasota, FL) and resistance measurements for blank inserts were subtracted. Cultures were determined to be confluent once TER plateaued. Confluent cultures were normalized for 48 hrs in MEM + 0.5% FBS. Prior to the permeability assay, cultures were equilibrated in physiologic Ringer’s solution for 30 minutes. Papp to 75 μg/ml sodium fluorescein was measured by apical sample fluorophotometer counts over 120 minutes following addition to the basolateral chamber. Papp was calculated in cm/s as apical counts per time interval divided by basolateral counts per volume times the area of the transwell aperture. Following the assay, transwell membranes were removed, cells were fixed, nuclei were stained with DAPI, and photographed with a fluorescence microscope to count ECD.
Papp and TER data was grouped by ECD into 3 categories (category [mean ± SD, n]): low, 1000-1499 cells/mm2 (1195 ± 163, n = 12); mid, 1500-1999 cells/mm2 (1721 ± 132, n = 5); and high, 2000-2999 cells/mm2 (2250 ± 282, n = 11). Although groups had significantly different ECDs (One-Way ANOVA, p<0.0001), no significant differences were observed in TER measurements or Papp at 120 minutes. TER plateau values in ohms were: low ECD, 33.5 ± 8.4; mid ECD, 31.8 ± 13.9; and high ECD, 31.3 ± 10.9. Papp measurements were (in cm/s): low ECD, 9.07 ± 2.64 × 10-5; mid ECD, 8.47 ± 1.67 × 10-5; and high ECD, 9.54 ± 3.68 x 10-5.
Within the range of 1000-2999 cells/mm2, the endothelial monolayer demonstrates no significant change in TER or Papp. Despite the large range of ECD, monolayer barrier integrity persists. In humans, corneal edema is not seen until ECD falls below 1000 cells/mm2. This study supports that observation and emphasizes the critical role of maintaining junctional integrity.
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