Heparin administration induced MMP 9 expression. (A) RT-PCR Southern blot analysis of MMP 9 mRNA. PCR products were subjected to Southern blot analyses with α-[³²P] dCTP–labeled cDNA probes. Specific radioactivity was normalized to the expression of β-actin (B-C). Twenty-four hours after MCAO, MMP 9 mRNA levels were increased in the ischemic brains of WT and tPA KO mice. MMP 9 mRNA levels were exacerbated significantly by heparin administration in WT mice but not in tPA KO mice. Values are means ± SD (n = 3). ^*P < .05, ^**P < .01, #P < .05, and ##P < .01 compared with contralateral hemispheres. (D) Immunofluorescence staining shows MMP 9 expression was observed in the ischemic area, whereas more MMP 9 expression was detected in the border of the ischemic core (IC) at 48 hours after MCAO in heparin-treated WT mice. (E-J) Brain sections were double stained for MMP 9 (green; E, arrows) and endothelial (ENDO) cells (red; F, arrows) or MMP 9 (green; H, arrows) and microglial (MICRO) cells (red; I, arrows). Merged images from the ischemic border zone indicate the increased MMP 9 expression was localized in endothelial cells (G, arrows) and microglial cells (J, arrows). Scale bars, 300 μm (D), 50 μm (E-G), and 3 μm (H-J).