TJ alterations in BMVECs during coculture with activated monocytes. Endothelial cells are positive for VWF(A) and express occludin (B, arrow) at cell-cell contacts. Application of monocytes resulted in occludin relocalization to cytoplasm (C, arrow). Monocyte migration occurs across TJs in the BBB model. (D) Endothelial cells demonstrate a flat surface without gaps in control BBB constructs (without monocytes). Scanning electron microscopy revealed monocyte attachment to endothelial cell contacts (E, arrows) and their migration between endothelial-cell borders (F, arrow). Massive monocyte migration in response to MCP-1 resulted in focal gap formation of the endothelial monolayer (G). Original magnification (A-C) × 200; (insets, B-C) × 400; (D-E) × 4000; (F) × 7000; (G) × 1200. Images were visualized using a 20 ×/0.5 NA (A-C) or a 40 ×/0.75 NA (B-C, insets) objective and were acquired using a Color View II digital CCD camera.