Assessing the barrier function of the retinal pigment epithelium in adult mice using transepithelial electrical resistance measurements and quantitative immunohistochemistry.

Abstract

The retinal pigment epithelium (RPE) plays a crucial role in the homeostasis of the vertebrate retina as its tight junctions form the outer blood-retina barrier and regulate the movement of substances between the blood and the neural retina. However, the outer blood-retina barrier breaks down in many degenerative retinal diseases, likely due to oxidative stress. This leads to fluid accumulation and inflammation in the retina. As mouse models are important for studying degenerative retinal diseases, methods to assess the integrity of RPE tight junctions in the mouse are needed. In this study, we established a system to measure the transepithelial electrical resistance (TEER) in mouse RPE using an Ussing chamber. We validated the sensitivity of the TEER measurements by adding oxidative stress-related substances, such as lipopolysaccharide and interleukin-1β, to the apical chamber. We used the same substances, which are known to affect tight junction proteins, to study their effect on the morphological integrity of the hexagonal RPE array in a flat-mount preparation. Antibody stainings for zonula occludens-1, claudin-1, and connexin 43 revealed morphological aberrations with an increased number of abnormal intersections after incubation with interleukin-1β. To further quantify this effect, we devised a new method to measure the angular deviations from the hexagonal RPE cell array. In summary, our results show that TEER and quantitative immunohistochemistry effectively assess the barrier function in mouse RPE and allow analyzing mouse models for retinal degeneration in the future.