Assessment of Macrophage and Blood‐Brain Barrier Function in the Resilient C57BL/6 Mouse and Non‐Resilient Balb/c Mouse

Abstract

Macrophage activation disrupts the blood‐brain barrier (BBB) in neurological and psychological diseases. However, the contribution of individual differences in macrophage and BBB phenotype in people’s susceptibility to these illnesses is largely unknown. We proposed to use a mouse model of psychological resilience to understand the role of macrophage and BBB function in disease susceptibility. In this model, the adoptive transfer of macrophages between Balb/c and C57BL/6 mice confers susceptibility or resistance, respectively, to development of depressive behaviors, thus highlighting the role of individual differences in macrophage function in brain‐related disorders.Our objective was to determine BBB function (permeability of brain microvascular endothelial cell (BMEC) monolayer), BBB response to endotoxin challenge (E. colilipopolysaccharide (LPS)), and macrophage activation (via LPS) in C57BL/6 mice and Balb/c mice. We hypothesized that 1) Balb/c BMEC monolayer will be innately more permeable than C57BL/6 BMEC monolayer, 2) Balb/c BMECs will be more sensitive to LPS, and 3) LPS‐activated bone‐marrow derived macrophages (BMMs) of Balb/c mice will induce greater disruption of BMECs than C57BL/6 BMMs.BMECs derived from C57BL/6 and Balb/c mice were seeded on 96‐well plates outfitted with electrodes to measure transendothelial electrical resistance (TEER) by Electric Cell‐substrate Impedance Sensing (Applied Biophysics, Inc.). After a stable monolayer was established, cells were treated with LPS (0, 25, 50, 75 or 100 ng/mL), or media conditioned by Balb/c or C57BL/6 BMMs plated at a density of 20,000 cells/cm2. BMMs were treated with 100 ng/mL of LPS (activated) or plain media (non‐activated). After 24 hours, the media was exchanged with fresh plain media, and collected after an additional 24 hours. Experiments were performed on three different days to obtain N=3/group.C57BL/6 BMEC monolayers displayed higher maximal TEER than Balb/c BMEC monolayer (24684 ± 1291 versus 19950 ± 1462 ohms cm2), and were significantly more sensitive to the TEER‐reducing effects of LPS after 12 hours of LPS exposure (FStrain(1, 20) = 7.4, P = 0.01). Media conditioned with LPS‐activated Balb/c BMMs induced significant disruption of C57BL/6 BMECs for up to 24 hours (FStrain(1,20) = 18.9, P = 0.0003), but had no effect on Balb/c BMECs. Media conditioned with non‐activated Balb/c BMMs, or activated or non‐activated C57BL/6 BMMs had no effect on TEER of either types of BMECs.Our study demonstrated that C57BL/6 mice have a less permeable BBB that is more sensitive to endotoxin challenge compared to BBB of Balb/c mice. C57BL/6 mice also possess macrophages that, when activated, are less capable of inducing BBB leakiness compared to Balb/c mice. The innate differences in macrophage and BBB function between the resilient C57BL/6 mouse and the non‐resilient Balb/c mouse likely play a role in their susceptibility to neurological/psychological disorders. We postulate that people’s susceptibility to brain‐related disorders is partly due to the interaction of their genetically‐determined BBB and macrophage phenotypes.Support or Funding InformationThe views expressed in this abstract are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense, or the U.S. Government.