A Novel Model for the Study of the Outer Blood-Retinal Barrier

Abstract

Purpose:: Current models of the outer blood-retinal barrier (oBRB) use cultured retinal pigment epithelial (RPE) cells to measure transepithelial electrical resistance (TEER). The integrity of the RPE and it's underlying Bruch's membrane is important in diseases such as age-related macular degeneration (AMD). However, current techniques do not allow measurements of TEER in physiologically intact tissues, such as in the RPE-choroidal complex. Here we establish a novel ex-vivo technique to study the oBRB using a modified Ussing chamber.

Methods:: The sclera and retina of six month old Brown Norway (BN) rats were micro-surgically removed and the RPE-choroidal complex was mounted into a modified Ussing chamber. This is a two half chamber system that was originally developed to measure ion transport and diffusion across biological membranes, using a current and a voltage passing pair of electrodes. Each half chamber was filled with DMEM which was kept at 35 ºC, and gassed with 5% CO₂, balanced N₂. A bipolar pulse (duration of 0.5 sec, 5 mV) was applied every 20-30 seconds, while the electrical current through the tissue was recorded. The recorded signal was averaged over a period of 10 minutes, and the noise was eliminated by averaging the single pulses over time. The unit area resistance, given in Ω·cm² was calculated using Ohm's law, followed by multiplication with the tissue's respective surface area. Integrity of the RPE layer was examined using immunohistochemistry staining for actin filaments (F-actin) and nuclei.

Results:: Our experiments show that this novel technique allows the measurement of TEER in the intact rat RPE-choroidal complex. Resistances of 123.62 ±40.62 Ω·cm² are measured in six month old Brown Norway rats, varying from 70 - 174 Ω·cm² (n=5).Averages were derived from the highest values per animal, while n represents the number of animals. Values that showed a constant resistance over a period of 12 minutes or longer (up to 50 minutes) were included. Post measurements, actin and nuclear staining confirmed the integrity or the rupture of the tissue and therefore contributed to the inclusion or exclusion of the data, respectively. These criteria lead to the exclusion of 11 out of 16 measured values.

Conclusions:: Our measured baseline TEER values are comparable with those previously reported in cultured ARPE-19 cells, suggesting that our technique may be a useful complement to the existing in vitro techniques.This novel technique can be used for the study of age- or disease-related changes to the oBRB.

Keywords: retinal pigment epithelium • electrophysiology: non-clinical • Bruch's membrane