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M. I. Melhorn, H.-G. Yu, A. S. Schering, D. Sun, S. Nakao, K. L. Thomas, J. W. Miller, E. S. Gragoudas, A. Hafezi-Moghadam; Impact of IL-1β on Transepithelial Electrical Resistance on Rodent RPE/Choroid Explants. Invest. Ophthalmol. Vis. Sci. 2008;49(13):4762. doi: https://doi.org/.
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Our recently introduced ex-vivo technique to study the outer blood-retinal barrier (oBRB) utilizes the intact retinal pigment epithelial (RPE)-choroidal tissue instead of cultured RPE cells for transepithelial electrical resistance (TEER) measurements. The role of inflammatory and angiogenic cytokines in age-related ocular diseases is commonly accepted, however, the impact of these molecules on the oBRB is unknown. Here we investigate the role of IL-1β in intact rat RPE-choroidal complex explants upon application to the solutions bathing the apical or basolateral membranes.
The sclera and retina of 2-6 month old Brown Norway (BN) rats were micro-surgically removed and the RPE-choroidal complex was mounted into a modified micro Ussing chamber. Each half chamber was filled with DMEM/F12, which was kept at 35 ± 0.5 ºC. A bipolar pulse (0.4 sec. duration, 5 mV) was applied every 30 seconds, while the electrical current was recorded. After the system reached stability, IL-1β was administered to the solutions bathing the apical or basolateral membrane. The unit area resistance or TEER in Ω·cm² was calculated using Ohm’s law, followed by multiplication with the tissue’s surface area. Integrity of the RPE layer was examined by staining for actin filaments, nuclei, and ZO-1.
An averaged resistance of 128 ± 30 Ω·cm² was measured in 2-6 months old Brown Norway rats. Individual values varied between 87-174 Ω·cm² (n=10). Apical application of 50 ng/ml IL-1β caused a significant reduction in TEER, within 30 minutes after administration. In contrast, basolateral administration of IL-1β did not lead to a comparable reduction in TEER in the same time frame. After measurements, actin-, nuclear-, and ZO-1 staining confirmed the integrity of the tissue and the tight junction molecules. The success rate of the reported results was approximately 60%.
Our experiments indicate that this new method allows the measurement of TEER in the intact rat RPE-choroidal complex ex-vivo. The measured baseline TEER values are comparable with those previously reported in our and other groups. The specific differences between the results of apical and basal IL-1β administration suggest a functional polarity in response to inflammatory mediators.
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