(A) Confocal microscopy images of the ischemic middle cerebral artery (MCA), cerebral micro-vessels (Mic.V), and surrounding brain tissue (Brain) immunofluorescently labeled for MMP-9 (a-f) or TIMP-1 (g-l). Images represent the vehicle control group (a, g), MCAO plus vehicle group (b, h), MCAO plus U0126 starting at 0 hours (c, i), 6 hours (d, j), or 12 hours (e, k) groups, and the negative control group (f, l). There was a significant increase in the MMP-9 protein level in the smooth muscle cell layer of ischemic vessels (MCA and Mic.V) as compared with vessels from the vehicle control group. TIMP-1 expression was upregulated in smooth muscle cells and in the proximity of the adventitia layer of ischemic vessels as compared to control vessels. Treatment with U0126, starting at zero and 6 hours, but not at 12 hours, after occlusion prevented the increase in MMP-9 and TIMP-1 protein expression. There was a slight increase in MMP-9 protein expression in ischemic brain tissue and in astrocytes around the vessels as compared to control and U0126-treated brain tissue. For TIMP-1, was no difference in protein expression in control brain tissue, in ischemic brain tissue, or tissue from animals treated with U0126. Scale bar, 50 μm. (B, C) Bar graphs showing the fluorescence intensity for MMP-9 and TIMP-1 in the MCA and micro-vessels. There was a significant increase in MMP-9 and TIMP-1 protein expression in MCAO animals as compared to control animals; this increase was prevented with U0126 treatment starting at zero and 6 hours, but not 12 hours, post MCA. Data are presented as the mean percentage relative to control ± SEM.; n = 5. *P < 0.05, **P < 0.01.