Figure 5

In vivo effect of AM9928 on transmigration of GFP-4T1-BrM5 cells across the BBB and on BMEC-Tight Junctions expression. (a) In vivo analysis of the BBB permeability: following injection of Evans blue, a statistically significant increase of the dye was found in the hippocampal region in mice administered with GFP-4T1-BrM5 cells and treated with vehicle control, but not in mice treated with AM9928. The level of dye in the control WT mice is shown also for comparison between the groups. This is a representative experiment of three separate experiments. The error bars indicate standard deviations. *p < 0.05 as compared to WT mice control. (b) Immunodetection of tight junctions in normal brain CD31 positive microvasculature (BMECs). The normal brain microvasculature was immunoassayed with CD31 antibody (1:500 dilution) as a marker for BMECs, using FITC- conjugated secondary antibody. Brain sections were immunoassayed for ZO-1(1:500 dilution) and Claudin-5 (1:500 dilution) using fluorescence Texas Red secondary antibody to detect ZO-1 and Claudin-5 expression in normal mouse brain sections. Arrows show the intact ZO-1(red) and Claudin-5 (red) structures, as indicated. Brain nuclei were counterstained with DAPI (blue). These are representative images of over 50 fields examined in three independent experiments. Scale bar = 20 μm. (c) In vivo effects of GFP-4T1-BrM5 tumor cells on the BBB integrity and on tight junctions’ structures: upper panel: the tumor cells were detected by pan-cytokeratin (Pan-CK) antibody (1:500 dilution) as primary antibody and fluorescence Texas red was used as a secondary antibody to detect the tumor cells in brain sections of mice administered with GFP-4T1-BrM5 cells. The brain microvasculature was immunoassayed with CD31 antibody (1:500 dilution) to detect BMECs (red). The arrows indicate the tumor cells or the CD31 BMECs as indicated. These are representative images of over 50 fields examined in three independent experiments. Magnification—× 40; scale bar-10 μm. Middle and Lower Panels: Immunodetection of tight junctions in brain tumors: Expression of Claudin-5 (middle panel) (green) and ZO-1 (lower panel) (green) in tumor brain sections were performed by immunostaining using FITC-conjugated secondary antibody as indicated. Detection of GFP-4T1-BrM5 cells in vivo was performed by immunostaining with Pan-CK antibody (red). Co-immunostaining of the tumor cells with Claudin-5 or with ZO-1 are shown in the merged figures. Brain nuclei were counterstained with DAPI (blue). These are representative images of over 50 fields examined from three independent experiments. The magnification—× 40; scale bar-10 μm.