FIGURE 4

Intracerebral (IC) administration of monocyte chemoattractant protein-1 (MCP-1) alters tight junction (TJ) complex proteins (in vivo study). (A) Mice received a single IC injection of MCP-1 (25 g) and were then killed at 6, 12, 24, and 48 h, or they received a continuous infusion of 5 g/h for 3 days or 2.5 g/h for 7 days. At killing, brain samples were processed for Western blot of TJ proteins (occludin, claudin-5, ZO-1, and ZO-2). GAPDH levels were also examined as a control. Representative Western blots from the brain ipsi- and contralateral to the site of injection are shown. Quantitative analysis (bar graphs of TJ protein/GAPDH ratios) of such blots showed a time-dependent decrease in the brain levels of each of the TJ proteins after MCP-1 treatment. Values are meanss.d. (B) Representative immunohistochemistry of altered TJ proteins in brain endothelial cells of mice after prolonged exposure to MCP-1 (5 g/h; intracerebral) for 3 days. Fluorescein isothiocyanate (FITC)-albumin was injected intravenously 1 h before the mouse was killed. In the contralateral hemisphere, FITC-albumin remained within the blood vessels and those vessels were all positive for occludin, claudin-5, ZO-1, and ZO-2 (adjacent FITC-albumin and TJ images are from the same area). In contrast, in the ipsilateral brain, there is extravasation of FITC-albumin and a loss of TJ proteins. Arrowheads point to blood vessels filled with FITC-albumin; Arrows point to TJ protein. Scale bar 80 m.