May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Impact of IL-1β on Transepithelial Electrical Resistance on Rodent RPE/Choroid Explants
Author Affiliations & Notes
  • M. I. Melhorn
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
    Institute for Molecular Biology and Bioinformatics, Charité, Campus Benjamin Franklin, Freie Universität Berlin, Berlin, Germany
  • H.-G. Yu
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • A. S. Schering
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • D. Sun
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • S. Nakao
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • K. L. Thomas
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • J. W. Miller
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • E. S. Gragoudas
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • A. Hafezi-Moghadam
    Ophthalmology, Massachusetts Eye & Ear Infirmary and Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  M.I. Melhorn, None; H. Yu, None; A.S. Schering, None; D. Sun, None; S. Nakao, None; K.L. Thomas, None; J.W. Miller, None; E.S. Gragoudas, None; A. Hafezi-Moghadam, None.
  • Footnotes
    Support  NIH grant AI050775 to A.H.-M., NEI core grant EY14104, Massachusetts Lions Foundation and Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 4762. doi:https://doi.org/
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : 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.

Methods: : 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.

Results: : 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%.

Conclusions: : 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.

Keywords: retinal pigment epithelium • electrophysiology: non-clinical • immune tolerance/privilege 
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